Methods and compositions for the specific inhibition of KRAS by asymmetric double-stranded RNA

ABSTRACT

This invention relates to compounds, compositions, and methods useful for reducing KRAS target RNA and protein levels via use of Dicer substrate siRNA (DsiRNA) agents possessing asymmetric end structures.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to, and the benefit under 35U.S.C. §119(e) of, the following applications: U.S. provisional patentapplication No. 61/166,559, filed Apr. 3, 2009, now expired; U.S.provisional patent application No. 61/166,578, filed Apr. 3, 2009, nowexpired; U.S. provisional patent application No. 61/174,279, filed Apr.30, 2009, now expired; U.S. provisional patent application No.61/174,306, filed Apr. 30, 2009, now expired; U.S. provisional patentapplication No. 61/257,810, filed Nov. 3, 2009, now expired; and U.S.provisional patent application No. 61/257,820, filed Nov. 3, 2009, nowexpired. The present application also claims priority to the followingapplications: U.S. patent application Ser. No. 12/704,256, filed Feb.11, 2010, published as US 2010-0249214 A1, now abandoned; U.S. patentapplication Ser. No. 12/642,371, filed Dec. 18, 2009, published as US2010-0173974 A1; U.S. patent application Ser. No. 12/642,404, filed Dec.18, 2009, published as US 2010-0202442 A1; U.S. patent application Ser.No. 12/642,264, filed Dec. 18, 2009, published as US 2010-0173973 A1,now abandoned; and PCT application No. PCT/US2009/005214, filed Sep. 17,2009, published as WO 2010/033225. The present application claimspriority to, and the benefit under 35 U.S.C. §119(e) of, the followingapplications: U.S. provisional patent application No. 61/183,815, filedJun. 3, 2009, now expired; U.S. provisional patent application No.61/183,818, filed Jun. 3, 2009, now expired; U.S. provisional patentapplication No. 61/184,735, filed Jun. 5, 2009, now expired; U.S.provisional patent application No. 61/285,925, filed Dec. 11, 2009, nowexpired; and U.S. provisional patent application No. 61/309,266, filedMar. 1, 2010, now expired. The entire teachings of the aboveapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of KRAS gene expression and/oractivity. The present invention is also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in KRAS gene expression pathways or other cellular processesthat mediate the maintenance or development of such traits, diseases andconditions. Specifically, the invention relates to small nucleic acidmolecules that are capable of being processed by the Dicer enzyme, suchas Dicer substrate siRNAs (DsiRNAs) capable of mediating RNAinterference (RNAi) against KRAS gene expression. Such anti-KRAS DsiRNAsare useful, for example, in providing compositions for treatment oftraits, diseases and conditions that can respond to modulation of KRASin a subject, such as cancer and/or other proliferative diseases,disorders, or conditions.

BACKGROUND OF THE INVENTION

Disregulated Ras signaling can lead to tumor growth and metastasis(Goodsell D S. Oncologist 4: 263-4). It is estimated that 20-25% of allhuman tumors contain activating mutations in Ras; and in specific tumortypes, this figure can be as high as 90% (Downward J. Nat Rev Cancer, 3:11-22). Accordingly, members of the Ras gene family are attractivemolecular targets for cancer therapeutic design.

The three human RAS genes encode highly related 188 to 189 amino acidproteins, designated H-Ras, N-Ras and K-Ras4A (KRAS isoform a) andK-Ras4B (KRAS isoform b; the two KRas proteins arise from alternativegene splicing). Ras proteins function as binary molecular switches thatcontrol intracellular signaling networks. Ras-regulated signal pathwayscontrol such processes as actin cytoskeletal integrity, proliferation,differentiation, cell adhesion, apoptosis, and cell migration. Ras andRas-related proteins are often deregulated in cancers, leading toincreased invasion and metastasis, and decreased apoptosis. Rasactivates a number of pathways but an especially important one fortumorigenesis appears to be the mitogen-activated protein (MAP) kinases,which themselves transmit signals downstream to other protein kinasesand gene regulatory proteins (Lodish et al. Molecular Cell Biology (4thed.). San Francisco: W.H. Freeman, Chapter 25, “Cancer”).

Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35nucleotides have been described as effective inhibitors of target geneexpression in mammalian cells (Rossi et al., U.S. Patent ApplicationNos. 2005/0244858 and US 2005/0277610). dsRNA agents of such length arebelieved to be processed by the Dicer enzyme of the RNA interference(RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA”(“DsiRNA”) agents. Additional modified structures of DsiRNA agents werepreviously described (Rossi et al., U.S. Patent Application No.2007/0265220).

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions that contain doublestranded RNA (“dsRNA”), and methods for preparing them. The dsRNAs ofthe invention are capable of reducing the expression of a target KRasgene in a cell, either in vitro or in a mammalian subject. Moreparticularly, the invention is directed to preferred Dicer substratesiRNAs (“DsiRNAs”) with structures and modification patterns that areoptimized to act as effective and highly potent KRAS inhibitory agents,optionally possessing extended duration of inhibitory effect. A majorityof such DsiRNAs possess target-specific inhibitory potencies andefficacies that are surprisingly enhanced relative to 21 nucleotidesiRNAs directed against the same target RNA. While not intending to bebound by theory, such enhanced activity likely reflects an advantageinherent in DsiRNA agents engaging the RNAi pathway at a point in thepathway that is upstream of the point at which shorter siRNA agentsengage the RNAi pathway.

In one aspect, the invention provides an isolated double strandedribonucleic acid (dsRNA) comprising first and second nucleic acidstrands and a duplex region of at least 25 base pairs, wherein thesecond strand of said dsRNA comprises 1-5 single-stranded nucleotides atits 3′ terminus, wherein the second oligonucleotide strand issufficiently complementary to a target KRAS cDNA sequence selected fromthe group consisting of SEQ ID NOs: 141-186 along at least 19nucleotides and at most 35 nucleotides of the second oligonucleotidestrand length to reduce KRAS target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell.

In one embodiment, starting from the first nucleotide (position 1) atthe 3′ terminus of the first oligonucleotide strand, position 1, 2and/or 3 is substituted with a modified nucleotide. In certainembodiments, the modified nucleotide residue of the 3′ terminus of thefirst strand is a deoxyribonucleotide, an acyclonucleotide or afluorescent molecule. In a related embodiment, position 1 of the 3′terminus of the first oligonucleotide strand is a deoxyribonucleotide.

In an additional embodiment, the 3′ terminus of the first strand and the5′ terminus of the second strand form a blunt end.

In another embodiment, the first strand is 25 nucleotides in length andthe second strand is 27 nucleotides in length.

In one embodiment, the second strand includes a sequence of SEQ ID NOs:11-50 and 135-140.

In another embodiment, the first strand includes a sequence of SEQ IDNOs: 5, 8 and 91-134.

In an additional embodiment, the dsRNA includes a pair of firststrand/second strand sequences that is SEQ ID NO: 5/SEQ ID NO: 14, SEQID NO: 8/SEQ ID NO: 43, SEQ ID NO: 132/SEQ ID NO: 138, SEQ ID NO: 91/SEQID NO: 11, SEQ ID NO: 129/SEQ ID NO: 135, SEQ ID NO: 92/SEQ ID NO: 12,SEQ ID NO: 130/SEQ ID NO: 136, SEQ ID NO: 93/SEQ ID NO: 13, SEQ ID NO:131/SEQ ID NO: 137, SEQ ID NO: 94/SEQ ID NO: 15, SEQ ID NO: 133/SEQ IDNO: 139, SEQ ID NO: 95/SEQ ID NO: 16, SEQ ID NO: 96/SEQ ID NO: 17, SEQID NO: 97/SEQ ID NO: 18, SEQ ID NO: 98/SEQ ID NO: 19, SEQ ID NO: 99/SEQID NO: 20, SEQ ID NO: 100/SEQ ID NO: 21, SEQ ID NO: 101/SEQ ID NO: 22,SEQ ID NO: 102/SEQ ID NO: 23, SEQ ID NO: 103/SEQ ID NO: 24, SEQ ID NO:104/SEQ ID NO: 25, SEQ ID NO: 105/SEQ ID NO: 26, SEQ ID NO: 106/SEQ IDNO: 27, SEQ ID NO: 107/SEQ ID NO: 28, SEQ ID NO: 108/SEQ ID NO: 29, SEQID NO: 109/SEQ ID NO: 30, SEQ ID NO: 110/SEQ ID NO: 31, SEQ ID NO:111/SEQ ID NO: 32, SEQ ID NO: 112/SEQ ID NO: 33, SEQ ID NO: 113/SEQ IDNO: 34, SEQ ID NO: 114/SEQ ID NO: 35, SEQ ID NO: 115/SEQ ID NO: 36, SEQID NO: 116/SEQ ID NO: 37, SEQ ID NO: 117/SEQ ID NO: 38, SEQ ID NO:118/SEQ ID NO: 39, SEQ ID NO: 119/SEQ ID NO: 40, SEQ ID NO: 134/SEQ IDNO: 140, SEQ ID NO: 120/SEQ ID NO: 41, SEQ ID NO: 121/SEQ ID NO: 42, SEQID NO: 122/SEQ ID NO: 44, SEQ ID NO: 123/SEQ ID NO: 45, SEQ ID NO:124/SEQ ID NO: 46, SEQ ID NO: 125/SEQ ID NO: 47, SEQ ID NO: 126/SEQ IDNO: 48, SEQ ID NO: 127/SEQ ID NO: 49 or SEQ ID NO: 128/SEQ ID NO: 50.

In one embodiment, each of the first and the second strands has a lengthwhich is at least 26 nucleotides.

In another embodiment, the nucleotides of the 3′ overhang include amodified nucleotide. Optionally, the modified nucleotide of the 3′overhang is a 2′-O-methyl ribonucleotide. In a related embodiment, allnucleotides of the 3′ overhang are modified nucleotides.

In an additional embodiment, one or both of the first and secondoligonucleotide strands includes a 5′ phosphate.

In another embodiment, the modified nucleotide residues of the dsRNA are2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O—(N-methlycarbamate).

In one embodiment, the 3′ overhang of the dsRNA is 1-3 nucleotides inlength. Optionally, the 3′ overhang is 1-2 nucleotides in length. In arelated embodiment, the 3′ overhang is two nucleotides in length and themodified nucleotide of the 3′ overhang is a 2′-O-methyl modifiedribonucleotide.

In a further embodiment, the second oligonucleotide strand, startingfrom the nucleotide residue of the second strand that is complementaryto the 5′ terminal nucleotide residue of the first oligonucleotidestrand, includes alternating modified and unmodified nucleotideresidues. In another embodiment, the second oligonucleotide strand,starting from the nucleotide residue of the second strand that iscomplementary to the 5′ terminal nucleotide residue of the firstoligonucleotide strand, includes unmodified nucleotide residues at allpositions from position 18 to the 5′ terminus of the secondoligonucleotide strand.

In another embodiment, each of the first and the second strands has alength which is at least 26 and at most 30 nucleotides.

In one embodiment, the dsRNA is cleaved endogenously in the cell byDicer.

In an additional embodiment, the amount of the isolated double strandednucleic acid sufficient to reduce expression of the target gene is 1nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50picomolar or less, 20 picomolar or less, 10 picomolar or less, 5picomolar or less, 2 picomolar or less or 1 picomolar or less in theenvironment of the cell.

In a further embodiment, the isolated dsRNA possesses greater potencythan isolated 21mer siRNAs directed to the identical at least 19nucleotides of the target KRAS cDNA in reducing target KRAS geneexpression when assayed in vitro in a mammalian cell at an effectiveconcentration in the environment of a cell of 1 nanomolar or less, 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, 10 picomolar or less, 5 picomolar or less, 2picomolar or less or 1 picomolar or less.

In another embodiment, the isolated dsRNA is sufficiently complementaryto the target KRAS cDNA sequence to reduce KRAS target gene expressionby at least 10%, at least 50%, at least 80-90%, at least 95%, at least98%, or at least 99% when the double stranded nucleic acid is introducedinto a mammalian cell.

In a further embodiment, the first and second strands are joined by achemical linker. In a related embodiment, the 3′ terminus of the firststrand and the 5′ terminus of the second strand are joined by a chemicallinker.

In one embodiment, a nucleotide of the second or first strand issubstituted with a modified nucleotide that directs the orientation ofDicer cleavage.

In another embodiment, the dsRNA has a modified nucleotide that is adeoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotideof 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine(3TC) and a monophosphate nucleotide of2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkylribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide,a 2′-fluoro ribonucleotide, or a locked nucleic acid.

In an additional embodiment, the dsRNA has a phosphonate, aphosphorothioate or a phosphotriesterphosphate backbone modification.

In one embodiment, the invention provides a method for reducingexpression of a target KRAS gene in a mammalian cell having contacting amammalian cell in vitro with an isolated dsRNA as described in an amountsufficient to reduce expression of a target KRAS gene in the cell.

In one embodiment, target KRAS gene expression is reduced by at least10%, at least 50%, or at least 80-90%. In another embodiment, targetKRAS mRNA levels are reduced at least 90% at least 8 days after the cellis contacted with the dsRNA. In a further embodiment, KRAS mRNA levelsare reduced by at least 70% at least 10 days after the cell is contactedwith the dsRNA.

In a further embodiment, the invention provides a method for reducingexpression of a target KRAS gene in a mammal by administering anisolated dsRNA as described to a mammal in an amount sufficient toreduce expression of a target KRAS gene in the mammal.

In one embodiment, the isolated dsRNA is administered at a dosage of 1microgram to 5 milligrams per kilogram of the mammal per day, 100micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams perkilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms perkilogram, 0.10 to 5 micrograms per kilogram, or 0.1 to 2.5 microgramsper kilogram.

In another embodiment, the isolated dsRNA possesses greater potency thanisolated 21 mer siRNAs directed to the identical at least 19 nucleotidesof the target KRAS cDNA in reducing target KRAS gene expression whenassayed in vitro in a mammalian cell at an effective concentration inthe environment of a cell of 1 nanomolar or less.

In an additional embodiment, the administering step includes intravenousinjection, intramuscular injection, intraperitoneal injection, infusion,subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical,oral or inhaled delivery.

In a further embodiment, the invention provides a method for selectivelyinhibiting the growth of a cell by contacting a cell with an amount ofan isolated dsRNA as described, in an amount sufficient to inhibit thegrowth of the cell.

In one embodiment, the cell is a tumor cell of a subject. Optionally,the cell is a tumor cell in vitro. In a related embodiment, the cell isa human cell.

In an additional embodiment, the invention provides a formulation whichincludes an isolated dsRNA as described, where the dsRNA is present inan amount effective to reduce target KRAS RNA levels when the dsRNA isintroduced into a mammalian cell in vitro by at least 10%, at least 50%or at least 80-90%, and where the dsRNA possesses greater potency thanisolated 21mer siRNAs directed to the identical at least 19 nucleotidesof the target KRAS cDNA in reducing target KRAS RNA levels when assayedin vitro in a mammalian cell at an effective concentration in theenvironment of a cell of 1 nanomolar or less.

In one embodiment, the effective amount is 1 nanomolar or less, 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, 10 picomolar or less, 5 picomolar or less, 2picomolar or less or 1 picomolar or less in the environment of the cell.

In another embodiment, the invention provides a formulation whichincludes an isolated dsRNA as described, where the dsRNA is present inan amount effective to reduce target RNA levels when the dsRNA isintroduced into a cell of a mammalian subject by at least 10%, at least50% or at least 80-90%, and where the dsRNA possesses greater potencythan isolated 21mer siRNAs directed to the identical at least 19nucleotides of the target KRAS cDNA in reducing target KRAS RNA levelswhen assayed in vitro in a mammalian cell at an effective concentrationin the environment of a cell of 1 nanomolar or less.

In one embodiment, the effective amount is a dosage of 1 microgram to 5milligrams per kilogram of the subject per day, 100 micrograms to 0.5milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.

In an additional embodiment, the invention provides a mammalian cellcontaining an isolated dsRNA as described.

Another embodiment of the invention provides a pharmaceuticalcomposition which includes an isolated dsRNA as described and apharmaceutically acceptable carrier. A further embodiment of theinvention provides a kit having an isolated dsRNA as described andinstructions for its use.

In an additional aspect, the invention provides a composition possessingKRAS inhibitory activity consisting essentially of an isolated doublestranded ribonucleic acid (dsRNA) comprising first and second nucleicacid strands and a duplex region of at least 25 base pairs, wherein thesecond strand of said dsRNA comprises 1-5 single-stranded nucleotides atits 3′ terminus, wherein the second oligonucleotide strand issufficiently complementary to a target KRAS cDNA sequence selected fromthe group consisting of SEQ ID NOs: 141-186 along at least 19nucleotides and at most 35 nucleotides of the second oligonucleotidestrand length to reduce KRAS target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell.

In another aspect, the invention provides an isolated double strandedribonucleic acid (dsRNA) comprising first and second nucleic acidstrands and a duplex region of at least 25 base pairs, wherein thesecond strand of said dsRNA comprises 1-5 single-stranded nucleotides atits 3′ terminus, wherein the second oligonucleotide strand issufficiently complementary to a target KRAS cDNA sequence selected fromthe group consisting of SEQ ID NOs: 1595-2297 and 3704-4406 along atleast 19 nucleotides and at most 35 nucleotides of the secondoligonucleotide strand length to reduce KRAS target gene expression whenthe double stranded nucleic acid is introduced into a mammalian cell.

In one embodiment, the second strand includes a sequence of SEQ ID NOs:189-891 and 2298-3000.

In another embodiment, the first strand includes a sequence of SEQ IDNOs: 892-1594 and 3001-3703.

In an additional embodiment, the dsRNA includes a DsiRNA agent selectedfrom the group consisting of DsiRNA agents shown in Tables 4-5.

In a further aspect, the invention provides a composition possessingKRAS inhibitory activity consisting essentially of an isolated doublestranded ribonucleic acid (dsRNA) comprising first and second nucleicacid strands and a duplex region of at least 25 base pairs, wherein thesecond strand of the dsRNA comprises 1-5 single-stranded nucleotides atits 3′ terminus, wherein the second oligonucleotide strand issufficiently complementary to a target KRAS cDNA sequence selected fromthe group consisting of SEQ ID NOs: 1595-2297 and 3704-4406 along atleast 19 nucleotides and at most 35 nucleotides of the secondoligonucleotide strand length to reduce KRAS target gene expression whenthe double stranded nucleic acid is introduced into a mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of exemplary DsiRNA agents targeting a sitein the KRAS RNA referred to herein as the “KRAS-355” target site. The25/27mer and 27/27mer DsiRNA agents (optionally, possessing a blunt/fraydesign) were tested for KRas inhibitory efficacy in comparison with the21mer siRNA constructs shown. UPPER case=unmodified RNA, lower case=DNA,Bold=mismatch base pair nucleotides; arrowheads indicate projected Dicerenzyme cleavage sites; dashed line indicates sense strand (top strand)sequences corresponding to the projected Argonaute 2 (Ago2) cleavagesite within the targeted KRas sequence.

FIG. 2 shows the anti-KRAS inhibitory efficacy of the agents depicted inFIG. 1, when transformed into cells in culture at 1 nanomolarconcentration. “Optimized”=25/27mer DsiRNA and 21mer siRNA of FIG. 1;Blunt/Blunt and Blunt/Fray agents are as indicated in FIG. 1;“Veh”=vehicle-treated control; “Un”=untreated control.

FIG. 3 shows the anti-KRAS inhibitory efficacy of the agents depicted inFIG. 1, when transformed into cells in culture at 100 picomolarconcentration. “Optimized”=25/27mer DsiRNA and 21mer siRNA of FIG. 1;Blunt/Blunt and Blunt/Fray agents are as indicated in FIG. 1;“Veh”=vehicle-treated control; “Un”=untreated control.

FIG. 4 shows the structures of exemplary DsiRNA agents targeting a sitein the KRAS RNA referred to herein as the “KRAS-940” target site. The25/27mer and 27/27mer DsiRNA agents (optionally, possessing a blunt/fraydesign) were tested for KRAS inhibitory efficacy in comparison with the21mer siRNA constructs shown. UPPER case=unmodified RNA, lower case=DNA,Bold=mismatch base pair nucleotides; arrowheads indicate projected Dicerenzyme cleavage sites; dashed line indicates sense strand (top strand)sequences corresponding to the projected Argonaute 2 (Ago2) cleavagesite within the targeted KRAS sequence.

FIG. 5 shows the anti-KRAS inhibitory efficacy of the agents depicted inFIG. 4, when transformed into cells in culture at 1 nanomolarconcentration. “Optimized”=25/27mer DsiRNA and 21mer siRNA of FIG. 4;Blunt/Blunt and Blunt/Fray agents are as indicated in FIG. 4;“Veh”=vehicle-treated control; “Un”=untreated control.

FIG. 6 shows the anti-KRAS inhibitory efficacy of the agents depicted inFIG. 4, when transformed into cells in culture at 100 picomolarconcentration. “Optimized”=25/27mer DsiRNA and 21mer siRNA of FIG. 4;Blunt/Blunt and Blunt/Fray agents are as indicated in FIG. 4;“Veh”=vehicle-treated control; “Un”=untreated control.

FIG. 7 shows a schematic comparison of tested DsiRNAs and their Dicerprocessing products, as compared to cognate 21 mer siRNAs directedagainst the same exact KRAS target sequence. 2′-O-methyl modificationsare indicated by open circles; shaded circles indicatedeoxyribonucleotides; shaded triangles indicate Dicer enzyme cleavagesites within a 25/27mer agent; open triangles indicate the position onthe antisense strand corresponding to the Argonaute 2 (Ago2) cleavagesites within the target RNA. Such sites are identical between the 40tested DsiRNA agents when compared to their cognate siRNA agents.

FIG. 8 shows a plot of comparative inhibitory efficacies betweenanti-KRAS DsiRNA 25/27mers and their cognate 21mer siRNAs. The plot isarranged in rank-order by % inhibition of KRAS target gene by the25/27mer DsiRNA agent. Arrows indicate the difference in efficacyobserved between DsiRNA agent and corresponding siRNA agent. Squaresindicate siRNA inhibitory results; circles indicate DsiRNA inhibitoryresults.

FIG. 9 shows a graph of inhibitory results arranged in rank-order byamount of difference in inhibitory effect between DsiRNA agents andtheir cognate siRNAs. Both DsiRNAs and siRNAs were tested at 24 hourspost-administration at both 5 nM (shaded squares) and 1 nM (shadedtriangles), and at 48 hours post administration at both 5 nM (opensquares) and 1 nM (open triangles). Shaded regions indicate agents forwhich a statistically significant difference was observed between DsiRNAand cognate siRNA. For 26 of the 40 tested anti-KRAS DsiRNA agents,statistically significant DsiRNA superiority was observed, as comparedto only six of forty siRNA agents that outperformed DsiRNA agents.

FIGS. 10 and 11 show IC-50 curves observed for indicated humanKRAS-targeting DsiRNAs.

FIGS. 12-21 show histograms of human KRAS inhibitory efficacies observedfor indicated DsiRNAs. “P1” indicates phase 1, while “P2” indicatesphase 2. In phase 1, DsiRNAs were tested at 1 nM in the environment ofHeLa cells. In phase 2, DsiRNAs were tested at 1 nM and at 0.1 nM (withduplicate experiments run at 0.1 nM) in the environment of HeLa cells.Individual bars represent average human KRAS levels observed intriplicate, with standard errors shown. Human KRAS levels werenormalized to HPRT and SFRS9 levels.

FIGS. 22-31 show histograms of mouse KRAS inhibitory efficacies observedfor indicated DsiRNAs. “P1” indicates phase 1, while “P2” indicatesphase 2. In phase 1, DsiRNAs were tested at 1 nM in the environment ofmouse Hepa 1-6 cells. In phase 2, DsiRNAs were tested at 1 nM and at 0.1nM (with duplicate experiments run at 0.1 nM) in the environment ofmouse Hepa 1-6 cells. Individual bars represent average mouse KRASlevels observed in triplicate, with standard errors shown. Mouse KRASlevels were normalized to HPRT and Rp123 levels.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions that contain doublestranded RNA (“dsRNA”), and methods for preparing them, that are capableof reducing the level and/or expression of the KRAS gene in vivo or invitro. One of the strands of the dsRNA contains a region of nucleotidesequence that has a length that ranges from 19 to 35 nucleotides thatcan direct the destruction and/or translational inhibition of thetargeted KRAS transcript.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

The present invention features one or more DsiRNA molecules that canmodulate (e.g., inhibit) KRAS expression. The DsiRNAs of the inventionoptionally can be used in combination with modulators of other genesand/or gene products associated with the maintenance or development ofdiseases or disorders associated with KRAS misregulation (e.g., tumorformation and/or growth, etc.). The DsiRNA agents of the inventionmodulate KRAS RNAs such as those corresponding to the cDNA sequencesreferred to by GenBank Accession Nos. NM_(—)033360 and NM_(—)004985,which are recited below and referred to herein generally as “KRAS.”

The below description of the various aspects and embodiments of theinvention is provided with reference to exemplary KRAS RNAs, generallyreferred to herein as KRAS. However, such reference is meant to beexemplary only and the various aspects and embodiments of the inventionare also directed to alternate KRAS RNAs, such as mutant KRAS RNAs oradditional KRAS splice variants. Certain aspects and embodiments arealso directed to other genes involved in KRAS pathways, including geneswhose misregulation acts in association with that of KRAS (or isaffected or affects KRAS regulation) to produce phenotypic effects thatmay be targeted for treatment (e.g., tumor formation and/or growth,etc.). Such additional genes can be targeted using DsiRNA and themethods described herein for use of KRAS targeting DsiRNAs. Thus, theinhibition and the effects of such inhibition of the other genes can beperformed as described herein.

The term “KRAS” refers to nucleic acid sequences encoding a KRasprotein, peptide, or polypeptide (e.g., KRAS transcripts, such as thesequences of KRAS Genbank Accession Nos. NM_(—)033360.2 andNM_(—)004985.3). In certain embodiments, the term “KRAS” is also meantto include other KRAS encoding sequence, such as other KRAS isoforms,mutant KRAS genes, splice variants of KRAS genes, and KRAS genepolymorphisms. The term “Kras” is used to refer to the polypeptide geneproduct of a KRAS gene/transript, e.g., a Kras protein, peptide, orpolypeptide, such as those encoded by KRAS Genbank Accession Nos.NM_(—)033360.2 and NM_(—)004985.3.

As used herein, a “KRAS-associated disease or disorder” refers to adisease or disorder known in the art to be associated with altered KRASexpression, level and/or activity. Notably, a “KRAS-associated diseaseor disorder” includes cancer and/or proliferative diseases, conditions,or disorders.

By “proliferative disease” or “cancer” as used herein is meant, adisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphdma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and other cancer or proliferative disease, condition,trait, genotype or phenotype that can respond to the modulation ofdisease related gene expression in a cell or tissue, alone or incombination with other therapies.

In certain embodiments, DsiRNA-mediated inhibition of a KRAS targetsequence is assessed. In such embodiments, KRAS RNA levels can beassessed by art-recognized methods (e.g., RT-PCR, Northern blot,expression array, etc.), optionally via comparison of KRAS levels in thepresence of an anti-KRAS DsiRNA of the invention relative to the absenceof such an anti-KRAS DsiRNA. In certain embodiments, KRAS levels in thepresence of an anti-KRAS DsiRNA are compared to those observed in thepresence of vehicle alone, in the presence of a DsiRNA directed againstan unrelated target RNA, or in the absence of any treatment. It is alsorecognized that levels of Kras protein can be assessed as indicative ofKRAS RNA levels and/or the extent to which a DsiRNA inhibits KRASexpression, thus art-recognized methods of assessing KRAS protein levels(e.g., Western blot, immunoprecipitation, other antibody-based methods,etc.) can also be employed to examine the inhibitory effect of a DsiRNAof the invention. An anti-KRAS DsiRNA of the invention is deemed topossess “KRAS inhibitory activity” if a statistically significantreduction in KRAS RNA or protein levels is seen when an anti-KRAS DsiRNAof the invention is administered to a system (e.g., cell-free in vitrosystem), cell, tissue or organism, as compared to an appropriatecontrol. The distribution of experimental values and the number ofreplicate assays performed will tend to dictate the parameters of whatlevels of reduction in KRAS RNA or protein (either as a % or in absoluteterms) is deemed statistically significant (as assessed by standardmethods of determining statistical significance known in the art).However, in certain embodiments, “KRAS inhibitory activity” is definedbased upon a % or absolute level of reduction in the level of KRAS in asystem, cell, tissue or organism. For example, in certain embodiments, aDsiRNA of the invention is deemed to possess KRAS inhibitory activity ifat least a 5% reduction or at least a 10% reduction in KRAS RNA isobserved in the presence of a DsiRNA of the invention relative to KRASlevels seen for a suitable control. (For example, in vivo KRAS levels ina tissue and/or subject can, in certain embodiments, be deemed to beinhibited by a DsiRNA agent of the invention if, e.g., a 5% or 10%reduction in KRAS levels is observed relative to a control.) In certainother embodiments, a DsiRNA of the invention is deemed to possess KRASinhibitory activity if KRAS RNA levels are observed to be reduced by atleast 15% relative to an appropriate control, by at least 20% relativeto an appropriate control, by at least 25% relative to an appropriatecontrol, by at least 30% relative to an appropriate control, by at least35% relative to an appropriate control, by at least 40% relative to anappropriate control, by at least 45% relative to an appropriate control,by at least 50% relative to an appropriate control, by at least 55%relative to an appropriate control, by at least 60% relative to anappropriate control, by at least 65% relative to an appropriate control,by at least 70% relative to an appropriate control, by at least 75%relative to an appropriate control, by at least 80% relative to anappropriate control, by at least 85% relative to an appropriate control,by at least 90% relative to an appropriate control, by at least 95%relative to an appropriate control, by at least 96% relative to anappropriate control, by at least 97% relative to an appropriate control,by at least 98% relative to an appropriate control or by at least 99%relative to an appropriate control. In some embodiments, completeinhibition of KRAS is required for a DsiRNA to be deemed to possess KRASinhibitory activity. In certain models (e.g., cell culture), a DsiRNA isdeemed to possess KRAS inhibitory activity if at least a 50% reductionin KRAS levels is observed relative to a suitable control. In certainother embodiments, a DsiRNA is deemed to possess KRAS inhibitoryactivity if at least an 80% reduction in KRAS levels is observedrelative to a suitable control.

KRAS inhibitory activity can also be evaluated over time (duration) andover concentration ranges (potency), with assessment of what constitutesa DsiRNA possessing KRAS inhibitory activity adjusted in accordance withconcentrations administered and duration of time followingadministration. Thus, in certain embodiments, a DsiRNA of the inventionis deemed to possess KRAS inhibitory activity if at least a 50%reduction in KRAS activity is observed at a duration of time of 2 hours,5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days or more after administration isobserved/persists. In additional embodiments, a DsiRNA of the inventionis deemed to be a potent KRAS inhibitory agent if KRAS inihibitoryactivity (e.g., in certain embodiments, at least 50% inhibition of KRAS)is observed at a concentration of 1 nM or less, 500 pM or less, 200 pMor less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5pM or less, 2 pM or less or even 1 pM or less in the environment of acell.

In certain embodiments, the phrase “consists essentially of” is used inreference to the anti-KRAS DsiRNAs of the invention. In some suchembodiments, “consists essentially of” refers to a composition thatcomprises a DsiRNA of the invention which possesse at least a certainlevel of KRAS inhibitory activity (e.g., at least 50% KRAS inhibitoryactivity) and that also comprises one or more additional componentsand/or modifications that do not significantly impact the KRASinhibitory activity of the DsiRNA. For example, in certain embodiments,a composition “consists essentially of” a DsiRNA of the invention wheremodifications of the DsiRNA of the invention and/or DsiRNA-associatedcomponents of the composition do not alter the KRAS inhibitory activity(optionally including potency or duration of KRAS inhibitory activity)by greater than 3%, greater than 5%, greater than 10%, greater than 15%,greater than 20%, greater than 25%, greater than 30%, greater than 35%,greater than 40%, greater than 45%, or greater than 50% relative to theDsiRNA of the invention in isolation. In certain embodiments, acomposition is deemed to consist essentially of a DsiRNA of theinvention even if more dramatic reduction of KRAS inhibitory activity(e.g., 80% reduction, 90% reduction, etc. in efficacy, duration and/orpotency) occurs in the presence of additional components ormodifications, yet where KRAS inhibitory activity is not significantlyelevated (e.g., observed levels of KRAS inhibitory activity are within10% those observed for the isolated DsiRNA of the invention) in thepresence of additional components and/or modifications.

As used herein, the term “nucleic acid” refers to deoxyribonucleotides,ribonucleotides, or modified nucleotides, and polymers thereof insingle- or double-stranded form. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

As used herein, “nucleotide” is used as recognized in the art to includethose with natural bases (standard), and modified bases well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Nucleotides generally comprise a base, sugarand a phosphate group. The nucleotides can be unmodified or modified atthe sugar, phosphate and/or base moiety, (also referred tointerchangeably as nucleotide analogs, modified nucleotides, non-naturalnucleotides, non-standard nucleotides and other; see, e.g., Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, hypoxanthine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

As used herein, “modified nucleotide” refers to a nucleotide that hasone or more modifications to the nucleoside, the nucleobase, pentosering, or phosphate group. For example, modified nucleotides excluderibonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate. Modifications include those naturallyoccurring that result from modification by enzymes that modifynucleotides, such as methyltransferases. Modified nucleotides alsoinclude synthetic or non-naturally occurring nucleotides. Synthetic ornon-naturally occurring modifications in nucleotides include those with2′ modifications, e.g., 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge,4′-(CH₂)₂—O-2′-bridge, 2′-LNA, and 2′-O—(N-methylcarbamate) or thosecomprising base analogs. In connection with 2′-modified nucleotides asdescribed for the present disclosure, by “amino” is meant 2′—NH₂ or2′-O—NH₂, which can be modified or unmodified. Such modified groups aredescribed, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 andMatulic-Adamic et al., U.S. Pat. No. 6,248,878.

In reference to the nucleic acid molecules of the present disclosure,modifications may exist upon these agents in patterns on one or bothstrands of the double stranded ribonucleic acid (dsRNA). As used herein,“alternating positions” refers to a pattern where every other nucleotideis a modified nucleotide or there is an unmodified nucleotide (e.g., anunmodified ribonucleotide) between every modified nucleotide over adefined length of a strand of the dsRNA (e.g., 5′-MNMNMN-3′;3′-MNMNMN-5′; where M is a modified nucleotide and N is an unmodifiednucleotide). The modification pattern starts from the first nucleotideposition at either the 5′ or 3′ terminus according to a positionnumbering convention, e.g., as described herein (in certain embodiments,position 1 is designated in reference to the terminal residue of astrand following a projected Dicer cleavage event of a DsiRNA agent ofthe invention; thus, position 1 does not always constitute a 3′ terminalor 5′ terminal residue of a pre-processed agent of the invention). Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but in certain embodiments includes at least4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7modified nucleotides, respectively. As used herein, “alternating pairsof positions” refers to a pattern where two consecutive modifiednucleotides are separated by two consecutive unmodified nucleotides overa defined length of a strand of the dsRNA (e.g., 5′-MMNNMMNNMMNN-3′;3′-MMNNMMNNMMNN-5′; where M is a modified nucleotide and N is anunmodified nucleotide). The modification pattern starts from the firstnucleotide position at either the 5′ or 3′ terminus according to aposition numbering convention such as those described herein. Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but preferably includes at least 8, 12, 16,20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14modified nucleotides, respectively. It is emphasized that the abovemodification patterns are exemplary and are not intended as limitationson the scope of the invention.

As used herein, “base analog” refers to a heterocyclic moiety which islocated at the 1′ position of a nucleotide sugar moiety in a modifiednucleotide that can be incorporated into a nucleic acid duplex (or theequivalent position in a nucleotide sugar moiety substitution that canbe incorporated into a nucleic acid duplex). In the dsRNAs of theinvention, a base analog is generally either a purine or pyrimidine baseexcluding the common bases guanine (G), cytosine (C), adenine (A),thymine (T), and uracil (U). Base analogs can duplex with other bases orbase analogs in dsRNAs. Base analogs include those useful in thecompounds and methods of the invention, e.g., those disclosed in U.S.Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent PublicationNo. 20080213891 to Manoharan, which are herein incorporated byreference. Non-limiting examples of bases include hypoxanthine (I),xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K),3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione)(P), iso-cytosine (iso-C), iso-guanine (iso-G),1-β-D-ribofuranosyl-(5-nitroindole),1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine,4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) andpyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S),2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole,4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl,7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl,tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer etal., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic AcidsResearch, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc.,119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324(1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Moraleset al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J.Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem.,63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci.,94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans.,1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1:1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632(2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri etal., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No.6,218,108.). Base analogs may also be a universal base.

As used herein, “universal base” refers to a heterocyclic moiety locatedat the 1′ position of a nucleotide sugar moiety in a modifiednucleotide, or the equivalent position in a nucleotide sugar moietysubstitution, that, when present in a nucleic acid duplex, can bepositioned opposite more than one type of base without altering thedouble helical structure (e.g., the structure of the phosphatebackbone). Additionally, the universal base does not destroy the abilityof the single stranded nucleic acid in which it resides to duplex to atarget nucleic acid. The ability of a single stranded nucleic acidcontaining a universal base to duplex a target nucleic can be assayed bymethods apparent to one in the art (e.g., UV absorbance, circulardichroism, gel shift, single stranded nuclease sensitivity, etc.).Additionally, conditions under which duplex formation is observed may bevaried to determine duplex stability or formation, e.g., temperature, asmelting temperature (Tm) correlates with the stability of nucleic acidduplexes. Compared to a reference single stranded nucleic acid that isexactly complementary to a target nucleic acid, the single strandednucleic acid containing a universal base forms a duplex with the targetnucleic acid that has a lower Tm than a duplex formed with thecomplementary nucleic acid. However, compared to a reference singlestranded nucleic acid in which the universal base has been replaced witha base to generate a single mismatch, the single stranded nucleic acidcontaining the universal base forms a duplex with the target nucleicacid that has a higher Tm than a duplex formed with the nucleic acidhaving the mismatched base.

Some universal bases are capable of base pairing by forming hydrogenbonds between the universal base and all of the bases guanine (G),cytosine (C), adenine (A), thymine (T), and uracil (U) under base pairforming conditions. A universal base is not a base that forms a basepair with only one single complementary base. In a duplex, a universalbase may form no hydrogen bonds, one hydrogen bond, or more than onehydrogen bond with each of G, C, A, T, and U opposite to it on theopposite strand of a duplex. Preferably, the universal bases does notinteract with the base opposite to it on the opposite strand of aduplex. In a duplex, base pairing between a universal base occurswithout altering the double helical structure of the phosphate backbone.A universal base may also interact with bases in adjacent nucleotides onthe same nucleic acid strand by stacking interactions. Such stackinginteractions stabilize the duplex, especially in situations where theuniversal base does not form any hydrogen bonds with the base positionedopposite to it on the opposite strand of the duplex. Non-limitingexamples of universal-binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (USPat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., Anacyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al.,3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNAsequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6;Loakes and Brown, 5-Nitroindole as an universal base analogue. NucleicAcids Res. 1994 Oct. 11; 22(20):4039-43).

As used herein, “loop” refers to a structure formed by a single strandof a nucleic acid, in which complementary regions that flank aparticular single stranded nucleotide region hybridize in a way that thesingle stranded nucleotide region between the complementary regions isexcluded from duplex formation or Watson-Crick base pairing. A loop is asingle stranded nucleotide region of any length. Examples of loopsinclude the unpaired nucleotides present in such structures as hairpins,stem loops, or extended loops.

As used herein, “extended loop” in the context of a dsRNA refers to asingle stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 basepairs or duplexes flanking the loop. In an extended loop, nucleotidesthat flank the loop on the 5′ side form a duplex with nucleotides thatflank the loop on the 3′ side. An extended loop may form a hairpin orstem loop.

As used herein, “tetraloop” in the context of a dsRNA refers to a loop(a single stranded region) consisting of four nucleotides that forms astable secondary structure that contributes to the stability of anadjacent Watson-Crick hybridized nucleotides. Without being limited totheory, a tetraloop may stabilize an adjacent Watson-Crick base pair bystacking interactions. In addition, interactions among the fournucleotides in a tetraloop include but are not limited tonon-Watson-Crick base pairing, stacking interactions, hydrogen bonding,and contact interactions (Cheong et al., Nature 1990 Aug. 16;346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4).A tetraloop confers an increase in the melting temperature (Tm) of anadjacent duplex that is higher than expected from a simple model loopsequence consisting of four random bases. For example, a tetraloop canconfer a melting temperature of at least 55° C. in 10 mM NaHPO₄ to ahairpin comprising a duplex of at least 2 base pairs in length. Atetraloop may contain ribonucleotides, deoxyribonucleotides, modifiednucleotides, and combinations thereof. Examples of RNA tetraloopsinclude the UNCG family of tetraloops (e.g., UUCG), the GNRA family oftetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., ProcNatl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., NucleicAcids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloopsinclude the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA))family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG)family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)).(Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002.; SHINJI et al.Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

As used herein, the term “siRNA” refers to a double stranded nucleicacid in which each strand comprises RNA, RNA analog(s) or RNA and DNA.The siRNA comprises between 19 and 23 nucleotides or comprises 21nucleotides. The siRNA typically has 2 bp overhangs on the 3′ ends ofeach strand such that the duplex region in the siRNA comprises 17-21nucleotides, or 19 nucleotides. Typically, the antisense strand of thesiRNA is sufficiently complementary with the target sequence of the KRASgene/RNA.

An anti-KRAS DsiRNA of the instant invention possesses strand lengths ofat least 25 nucleotides. Accordingly, an anti-KRAS DsiRNA contains oneoligonucleotide sequence, a first sequence, that is at least 25nucleotides in length and no longer thin 35 or up to 50 nucleotides.This sequence of RNA can be between 26 and 35, 26 and 34, 26 and 33, 26and 32, 26 and 31, 26 and 30, and 26 and 29 nucleotides in length. Thissequence can be 27 or 28 nucleotides in length or 27 nucleotides inlength. The second sequence of the DsiRNA agent can be a sequence thatanneals to the first sequence under biological conditions, such aswithin the cytoplasm of a eukaryotic cell. Generally, the secondoligonucleotide sequence will have at least 19 complementary base pairswith the first oligonucleotide sequence, more typically the secondoligonucleotide sequence will have 21 or more complementary base pairs,or 25 or more complementary base pairs with the first oligonucleotidesequence. In one embodiment, the second sequence is the same length asthe first sequence, and the DsiRNA agent is blunt ended. In anotherembodiment, the ends of the DsiRNA agent have one or more overhangs.

In certain embodiments, the first and second oligonucleotide sequencesof the DsiRNA agent exist on separate oligonucleotide strands that canbe and typically are chemically synthesized. In some embodiments, bothstrands are between 26 and 35 nucleotides in length. In otherembodiments, both strands are between 25 and 30 or 26 and 30 nucleotidesin length. In one embodiment, both strands are 27 nucleotides in length,are completely complementary and have blunt ends. In certain embodimentsof the instant invention, the first and second sequences of an anti-KRASDsiRNA exist on separate RNA oligonucleotides (strands). In oneembodiment, one or both oligonucleotide strands are capable of servingas a substrate for Dicer. In other embodiments, at least onemodification is present that promotes Dicer to bind to thedouble-stranded RNA structure in an orientation that maximizes thedouble-stranded RNA structure's effectiveness in inhibiting geneexpression. In certain embodiments of the instant invention, theanti-KRAS DsiRNA agent is comprised of two oligonucleotide strands ofdiffering lengths, with the anti-KRAS DsiRNA possessing a blunt end atthe 3′ terminus of a first strand (sense strand) and a 3′ overhang atthe 3′ terminus of a second strand (antisense strand). The DsiRNA canalso contain one or more deoxyribonucleic acid (DNA) base substitutions.

Suitable DsiRNA compositions that contain two separate oligonucleotidescan be chemically linked outside their annealing region by chemicallinking groups. Many suitable chemical linking groups are known in theart and can be used. Suitable groups will not block Dicer activity onthe DsiRNA and will not interfere with the directed destruction of theRNA transcribed from the target gene. Alternatively, the two separateoligonucleotides can be linked by a third oligonucleotide such that ahairpin structure is produced upon annealing of the two oligonucleotidesmaking up the DsiRNA composition. The hairpin structure will not blockDicer activity on the DsiRNA and will not interfere with the directeddestruction of the target RNA.

As used herein, a dsRNA, e.g., DsiRNA or siRNA, having a sequence“sufficiently complementary” to a target RNA or cDNA sequence (e.g.,KRAS mRNA) means that the dsRNA has a sequence sufficient to trigger thedestruction of the target RNA (where a cDNA sequence is recited, the RNAsequence corresponding to the recited cDNA sequence) by the RNAimachinery (e.g., the RISC complex) or process. The dsRNA molecule can bedesigned such that every residue of the antisense strand iscomplementary to a residue in the target molecule. Alternatively,substitutions can be made within the molecule to increase stabilityand/or enhance processing activity of said molecule. Substitutions canbe made within the strand or can be made to residues at the ends of thestrand. In certain embodiments, substitutions and/or modifications aremade at specific residues within a DsiRNA agent. Such substitutionsand/or modifications can include, e.g., deoxy-modifications at one ormore residues of positions 1, 2 and 3 when numbering from the 3′terminal position of the sense strand of a DsiRNA agent; andintroduction of 2′-O-alkyl (e.g., 2′-O-methyl) modifications at the 3′terminal residue of the antisense strand of DsiRNA agents, with suchmodifications also being performed at overhang positions of the 3′portion of the antisense strand and at alternating residues of theantisense strand of the DsiRNA that are included within the region of aDsiRNA agent that is processed to form an active siRNA agent. Thepreceding modifications are offered as exemplary, and are not intendedto be limiting in any manner. Further consideration of the structure ofpreferred DsiRNA agents, including further description of themodifications and substitutions that can be performed upon the anti-KRASDsiRNA agents of the instant invention, can be found below.

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or other manner thatallows for a duplex between oligonucleotide strands that arecomplementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 bases oneach strand are complementary or substantially complementary, such thatthe “duplex region” consists of 19 base pairs. The remaining base pairsmay, for example, exist as 5′ and 3′ overhangs. Further, within theduplex region, 100% complementarity is not required; substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to complementarity between the strands such thatthey are capable of annealing under biological conditions. Techniques toempirically determine if two strands are capable of annealing underbiological conditions are well know in the art. Alternatively, twostrands can be synthesized and added together under biologicalconditions to determine if they anneal to one another.

Single-stranded nucleic acids that base pair over a number of bases aresaid to “hybridize.” Hybridization is typically determined underphysiological or biologically relevant conditions (e.g., intracellular:pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion).Hybridization conditions generally contain a monovalent cation andbiologically acceptable buffer and may or may not contain a divalentcation, complex anions, e.g. gluconate from potassium gluconate,uncharged species such as sucrose, and inert polymers to reduce theactivity of water in the sample, e.g. PEG. Such conditions includeconditions under which base pairs can form.

Hybridization is measured by the temperature required to dissociatesingle stranded nucleic acids forming a duplex, i.e., (the meltingtemperature; Tm). Hybridization conditions are also conditions underwhich base pairs can form. Various conditions of stringency can be usedto determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger(1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507). Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Forexample, a hybridization determination buffer is shown in Table 1.

TABLE 1 To make 50 final conc. Vender Cat# Lot# m.w./Stock mL solutionNaCl 100 mM Sigma S-5150 41K8934 5M 1 mL KCl 80 mM Sigma P-9541 70K0002 74.55 0.298 g MgCl₂ 8 mM Sigma M-1028 120K8933 1M 0.4 mL sucrose 2% w/vFisher BP220-212 907105 342.3 1 g Tris-HCl 16 mM Fisher BP1757-500 124191M 0.8 mL NaH₂PO₄ 1 mM Sigma S-3193 52H-029515 120.0 0.006 g EDTA 0.02mM Sigma E-7889 110K89271 0.5M   2 μL H₂O Sigma W-4502 51K2359 to 50 mLpH = 7.0 adjust with HCl at 20° C.

Useful variations on hybridization conditions will be readily apparentto those skilled in the art. Hybridization techniques are well known tothose skilled in the art and are described, for example, in Benton andDavis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001); Berger and Kimmel(Antisense to Molecular Cloning Techniques, 1987, Academic Press, NewYork); and Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York.

As used herein, “oligonucleotide strand” is a single stranded nucleicacid molecule. An oligonucleotide may comprise ribonucleotides,deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2′modifications, synthetic base analogs, etc.) or combinations thereof.Such modified oligonucleotides can be preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases.

As used herein, the term “ribonucleotide” encompasses natural andsynthetic, unmodified and modified ribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between ribonucleotides in the oligonucleotide. As used herein,the term “ribonucleotide” specifically excludes a deoxyribonucleotide,which is a nucleotide possessing a single proton group at the 2′ ribosering position.

As used herein, the term “deoxyribonucleotide” encompasses natural andsynthetic, unmodified and modified deoxyribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between deoxyribonucleotide in the oligonucleotide. As usedherein, the term “deoxyribonucleotide” also includes a modifiedribonucleotide that does not permit Dicer cleavage of a dsRNA agent,e.g., a 2′-O-methyl ribonucleotide, a phosphorothioate-modifiedribonucleotide residue, etc., that does not permit Dicer cleavage tooccur at a bond of such a residue.

As used herein, the term “PS-NA” refers to a phosphorothioate-modifiednucleotide residue. The term “PS-NA” therefore encompasses bothphosphorothioate-modified ribonucleotides (“PS-RNAs”) andphosphorothioate-modified deoxyriibonucleotides (“PS-DNAs”).

As used herein, “Dicer” refers to an endoribonuclease in the RNase IIIfamily that cleaves a dsRNA or dsRNA-containing molecule, e.g.,double-stranded RNA (dsRNA) or pre-microRNA (miRNA), intodouble-stranded nucleic acid fragments 19-25 nucleotides long, usuallywith a two-base overhang on the 3′ end. With respect to the dsRNAs ofthe invention, the duplex formed by a dsRNA region of an agent of theinvention is recognized by Dicer and is a Dicer substrate on at leastone strand of the duplex. Dicer catalyzes the first step in the RNAinterference pathway, which consequently results in the degradation of atarget RNA. The protein sequence of human Dicer is provided at the NCBIdatabase under accession number NP_(—)085124, hereby incorporated byreference.

Dicer “cleavage” is determined as follows (e.g., see Collingwood et al.,Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNAduplexes (100 pmol) are incubated in 20 μL of 20 mM Tris pH 8.0, 200 mMNaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer(Stratagene, La Jolla, Calif.) at 37° C. for 18-24 hours. Samples aredesalted using a Performa SR 96-well plate (Edge Biosystems,Gaithersburg, Md.). Electrospray-ionization liquid chromatography massspectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment withDicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hailet al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur datasystem, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources, Auburn, Calif.). In this assay, Dicer cleavage occurswhere at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, oreven 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA,preferably 26-30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bpdsRNA, preferably, 21-23 bp dsRNA).

As used herein, “Dicer cleavage site” refers to the sites at which Dicercleaves a dsRNA (e.g., the dsRNA region of a DsiRNA agent of theinvention). Dicer contains two RNase III domains which typically cleaveboth the sense and antisense strands of a dsRNA. The average distancebetween the RNase III domains and the PAZ domain determines the lengthof the short double-stranded nucleic acid fragments it produces and thisdistance can vary (Macrae I, et al. (2006). “Structural basis fordouble-stranded RNA processing by Dicer”. Science 311 (5758): 195-8.).As shown in FIGS. 1 and 4, Dicer is projected to cleave certaindouble-stranded ribonucleic acids of the instant invention that possessan antisense strand having a 2 nucleotide 3′ overhang at a site betweenthe 21^(st) and 22^(nd) nucleotides removed from the 3′ terminus of theantisense strand, and at a corresponding site between the 21^(st) and22^(nd) nucleotides removed from the 5′ terminus of the sense strand.The projected and/or prevalent Dicer cleavage site(s) for dsRNAmolecules distinct from those depicted in FIGS. 1 and 4 may be similarlyidentified via art-recognized methods, including those described inMacrae et al. While the Dicer cleavage events depicted in FIGS. 1 and 4generate 21 nucleotide siRNAs, it is noted that Dicer cleavage of adsRNA (e.g., DsiRNA) can result in generation of Dicer-processed siRNAlengths of 19 to 23 nucleotides in length. Indeed, in certainembodiments, a double-stranded DNA region may be included within a dsRNAfor purpose of directing prevalent Dicer excision of a typicallynon-preferred 19mer or 20mer siRNA, rather than a 21mer.

As used herein, “overhang” refers to unpaired nucleotides, in thecontext of a duplex having one or more free ends at the 5′ terminus or3′ terminus of a dsRNA. In certain embodiments, the overhang is a 3′ or5′ overhang on the antisense strand or sense strand. In someembodiments, the overhang is a 3′ overhang having a length of betweenone and six nucleotides, optionally one to five, one to four, one tothree, one to two, two to six, two to five, two to four, two to three,three to six, three to five, three to four, four to six, four to five,five to six nucleotides, or one, two, three, four, five or sixnucleotides.

As used herein, the term “RNA processing” refers to processingactivities performed by components of the siRNA, miRNA or RNase Hpathways (e.g., Drosha, Dicer, Argonaute2 or other RISCendoribonucleases, and RNaseH), which are described in greater detailbelow (see “RNA Processing” section below). The term is explicitlydistinguished from the post-transcriptional processes of 5′ capping ofRNA and degradation of RNA via non-RISC- or non-RNase H-mediatedprocesses. Such “degradation” of an RNA can take several forms, e.g.deadenylation (removal of a 3′ poly(A) tail), and/or nuclease digestionof part or all of the body of the RNA by one or more of several endo- orexo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3,4/5), oligonucleotidase, etc.).

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of theDsiRNA agents of the instant invention contemplates the possibility ofusing such DsiRNA agents not only against target RNAs of KRAS possessingperfect complementarity with the presently described DsiRNA agents, butalso against target KRAS RNAs possessing sequences that are, e.g., only99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%,85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said DsiRNA agents.Similarly, it is contemplated that the presently described DsiRNA agentsof the instant invention might be readily altered by the skilled artisanto enhance the extent of complementarity between said DsiRNA agents anda target KRAS RNA, e.g., of a specific allelic variant of KRAS (e.g., anallele of enhanced therapeutic interest). Indeed, DsiRNA agent sequenceswith insertions, deletions, and single point mutations relative to thetarget KRAS sequence can also be effective for inhibition.Alternatively, DsiRNA agent sequences with nucleotide analogsubstitutions or insertions can be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions.times.100), optionally penalizing the score for the number ofgaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the DsiRNA antisense strand and theportion of the KRAS RNA sequence is preferred. Alternatively, the DsiRNAmay be defined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the KRAS RNA(e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.hybridization for 12-16 hours; followed by washing). Additionalpreferred hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length ofthe identical nucleotide sequences may be at least 10, 12, 15, 17, 20,22, 25, 27 or 30 bases.

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a DsiRNA moleculehaving complementarity to an antisense region of the DsiRNA molecule. Inaddition, the sense region of a DsiRNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a DsiRNAmolecule having complementarity to a target nucleic acid sequence. Inaddition, the antisense region of a DsiRNA molecule comprises a nucleicacid sequence having complementarity to a sense region of the DsiRNAmolecule.

As used herein, “antisense strand” refers to a single stranded nucleicacid molecule which has a sequence complementary to that of a targetRNA. When the antisense strand contains modified nucleotides with baseanalogs, it is not necessarily complementary over its entire length, butmust at least hybridize with a target RNA.

As used herein, “sense strand” refers to a single stranded nucleic acidmolecule which has a sequence complementary to that of an antisensestrand. When the antisense strand contains modified nucleotides withbase analogs, the sense strand need not be complementary over the entirelength of the antisense strand, but must at least duplex with theantisense strand.

As used herein, “guide strand” refers to a single stranded nucleic acidmolecule of a dsRNA or dsRNA-containing molecule, which has a sequencesufficiently complementary to that of a target RNA to result in RNAinterference. After cleavage of the dsRNA or dsRNA-containing moleculeby Dicer, a fragment of the guide strand remains associated with RISC,binds a target RNA as a component of the RISC complex, and promotescleavage of a target RNA by RISC. As used herein, the guide strand doesnot necessarily refer to a continuous single stranded nucleic acid andmay comprise a discontinuity, preferably at a site that is cleaved byDicer. A guide strand is an antisense strand.

As used herein, “passenger strand” refers to an oligonucleotide strandof a dsRNA or dsRNA-containing molecule, which has a sequence that iscomplementary to that of the guide strand. As used herein, the passengerstrand does not necessarily refer to a continuous single strandednucleic acid and may comprise a discontinuity, preferably at a site thatis cleaved by Dicer. A passenger strand is a sense strand.

By “target nucleic acid” is meant a nucleic acid sequence whoseexpression, level or activity is to be modulated. The target nucleicacid can be DNA or RNA. For agents that target KRAS, in certainembodiments target nucleic acid is KRAS RNA. KRAS RNA target sites canalso interchangeably be referenced by corresponding cDNA sequences.Levels of KRAS may also be targeted via targeting of upstream effectorsof KRAS, or the effects of modulated or misregulated KRAS may also bemodulated by targeting of molecules downstream of KRAS in the Rassignalling pathway.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, a DsiRNA moleculeof the invention comprises 19 to 30 (e.g., 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 or more) nucleotides that are complementary to oneor more target nucleic acid molecules or a portion thereof.

In one embodiment, DsiRNA molecules of the invention that down regulateor reduce KRAS gene expression are used for treating, preventing orreducing KRAS-related diseases or disorders (e.g., cancer) in a subjector organism.

In one embodiment of the present invention, each sequence of a DsiRNAmolecule of the invention is independently 25 to 35 nucleotides inlength, in specific embodiments 25, 26, 27, 28, 29, 30, 31, 32, 33, 34or 35 nucleotides in length. In another embodiment, the DsiRNA duplexesof the invention independently comprise 25 to 30 base pairs (e.g., 25,26, 27, 28, 29, or 30). In another embodiment, one or more strands ofthe DsiRNA molecule of the invention independently comprises 19 to 35nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34 or 35) that are complementary to a target (KRAS) nucleic acidmolecule. In certain embodiments, a DsiRNA molecule of the inventionpossesses a length of duplexed nucleotides between 25 and 34 nucleotidesin length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 nucleotides inlength; optionally, all such nucleotides base pair with cognatenucleotides of the opposite strand). (Exemplary DsiRNA molecules of theinvention are shown in FIGS. 1 and 4, and below.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell. Within certainaspects, the term “cell” refers specifically to mammalian cells, such ashuman cells, that contain one or more isolated dsRNA molecules of thepresent disclosure. In particular aspects, a cell processes dsRNAs ordsRNA-containing molecules resulting in RNA intereference of targetnucleic acids, and contains proteins and protein complexes required forRNAi, e.g., Dicer and RISC.

The DsiRNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in FIGS. 1 and 4, and the below exemplarystructures. Examples of such nucleic acid molecules consist essentiallyof sequences defined in these figures and exemplary structures.Furthermore, where such agents are modified in accordance with the belowdescription of modification patterning of DsiRNA agents, chemicallymodified forms of constructs described in FIGS. 1 and 4, and the belowexemplary structures can be used in all uses described for the DsiRNAagents of FIGS. 1 and 4, and the below exemplary structures.

In another aspect, the invention provides mammalian cells containing oneor more DsiRNA molecules of this invention. The one or more DsiRNAmolecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the DsiRNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the DsiRNA agents of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The phrase “pharmaceutically acceptable carrier” refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA compositions may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (seeKreuter, 1991). The polymeric materials which are formed from monomericand/or oligomeric precursors in the polymerization/nanoparticlegeneration step, are per se known from the prior art, as are themolecular weights and molecular weight distribution of the polymericmaterial which a person skilled in the field of manufacturingnanoparticles may suitably select in accordance with the usual skill.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isa control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent (e.g., DsiRNA) of theinvention into a cell or organism. In another embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined in a cell or organism, e.g., acontrol or normal cell or organism, exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a DsiRNA agent or avector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease or disorder, or symptoms of the disease or disorder. The term“treatment” or “treating” is also used herein in the context ofadministering agents prophylactically. The term “effective dose” or“effective dosage” is defined as an amount sufficient to achieve or atleast partially achieve the desired effect. The term “therapeuticallyeffective dose” is defined as an amount sufficient to cure or at leastpartially arrest the disease and its complications in a patient alreadysuffering from the disease. The term “patient” includes human and othermammalian subjects that receive either prophylactic or therapeutictreatment.

Structures of Anti-KRAS DsiRNA Agents

In certain embodiments, the anti-KRAS DsiRNA agents of the invention canhave the following structures:

In one such embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, and “D”=DNA. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand.

In an additional embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one related embodiment, theDsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. In afurther related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. The top strand is the sensestrand, and the bottom strand is the antisense strand. In anotherrelated embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one related embodiment, theDsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In another related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one related embodiment, theDsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In another related embodiment, the DsiRNAcomprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′430-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In a related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXX XXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNAmonomers. The top strand is the sense strand, and the bottom strand isthe antisense strand. In one related embodiment, the DsiRNA comprises:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In another related embodiment,the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group and “X”=2′-O-methyl RNA. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a further related embodiment, the DsiRNA comprises:

 5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

The above modification patterns can also be incorporated into, e.g., theextended DsiRNA structures and mismatch and/or frayed DsiRNA structuresdescribed below.

In another embodiment, the DsiRNA comprises strands having equal lengthspossessing 1-3 mismatched residues that serve to orient Dicer cleavage(specifically, one or more of positions 1, 2 or 3 on the first strand ofthe DsiRNA, when numbering from the 3′-terminal residue, are mismatchedwith corresponding residues of the 5′-terminal region on the secondstrand when first and second strands are annealed to one another). Anexemplary 27mer DsiRNA agent with two terminal mismatched residues isshown:

5′-pXXXXXXXXXXXXXXXXXXXXXXXXX^(M) ^(M) -3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXX_(M) _(Mp) -5′wherein “X”=RNA, “p”=a phosphate group, “M”=Nucleic acid residues (RNA,DNA or non-natural or modified nucleic acids) that do not base pair(hydrogen bond) with corresponding “M” residues of otherwisecomplementary strand when strands are annealed. Any of the residues ofsuch agents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown for aboveasymmetric agents, can also be used in the above “blunt/fray” DsiRNAagent. In one embodiment, the top strand is the sense strand, and thebottom strand is the antisense strand. Alternatively, the bottom strandis the sense strand and the top strand is the antisense strand.

In certain additional embodiments, the present invention providescompositions for RNA interference (RNAi) that possess one or more basepaired deoxyribonucleotides within a region of a double strandedribonucleic acid (dsRNA) that is positioned 3′ of a projected sensestrand Dicer cleavage site and correspondingly 5′ of a projectedantisense strand Dicer cleavage site. The compositions of the inventioncomprise a dsRNA which is a precursor molecule, i.e., the dsRNA of thepresent invention is processed in vivo to produce an active smallinterfering nucleic acid (siRNA). The dsRNA is processed by Dicer to anactive siRNA which is incorporated into RISC.

In certain embodiments, the DsiRNA agents of the invention can have thefollowing exemplary structures (noting that any of the followingexemplary structures can be combined, e.g., with the bottom strandmodification patterns of the above-described structures—in one specificexample, the bottom strand modification pattern shown in any of theabove structures is applied to the 27 most 3′ residues of the bottomstrand of any of the following structures; in another specific example,the bottom strand modification pattern shown in any of the abovestructures upon the 23 most 3′ residues of the bottom strand is appliedto the 23 most 3′ residues of the bottom strand of any of the followingstructures):

In one such embodiment, the DsiRNA comprises the following (an exemplary“right-extended”, “DNA extended” DsiRNA):

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)XX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In an additional embodiment, the DsiRNA comprises:

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises:

 5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)[X1/D1]_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)[X2/D2]_(N)ZZ-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA or RNA,and “N”=1 to 50 or more, but is optionally 1-8 or 1-10, where at leastone D1_(N) is present in the top strand and is base paired with acorresponding D2_(N) in the bottom strand. Optionally, D1_(N) andD1_(N+1) are base paired with corresponding D2_(N) and D2_(N+1); D1_(N),D1_(N+1) and D1_(N+2) are base paired with corresponding D2_(N),D1_(N+1) and D1_(N+2), etc. “N*”=0 to 15 or more, but is optionally 0,1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In the structures depicted herein, the 5′ end of either the sense strandor antisense strand can optionally comprise a phosphate group.

In another embodiment, the DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another). An exemplary DNA:DNA-extended DsiRNA agent with twoterminal mismatched residues is shown:

5′-XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N) ^(M) ^(M-3′)3′-XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N) _(MM-5′)                                             wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-8 or 1-10. “N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or6. Any of the residues of such agents can optionally be 2′-O-methyl RNAmonomers—alternating positioning of 2′-O-methyl RNA monomers thatcommences from the 3′-terminal residue of the bottom (second) strand, asshown for above asymmetric agents, can also be used in the above“blunt/fray” DsiRNA agent. In one embodiment, the top strand (firststrand) is the sense strand, and the bottom strand (second strand) isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. Modification andDNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In one embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsRNA structure. Anexemplary structure for such a molecule is shown:

 5′-XXXXXXXXXXXXXXXXXXXDDXX-3′ 3′-YXXXXXXXXXXXXXXXXXDDXXXX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. The above structureis modeled to force Dicer to cleave a minimum of a 21mer duplex as itsprimary post-processing form. In embodiments where the bottom strand ofthe above structure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand is likely to reduce off-targeteffects (as prior studies have shown a 2′-O-methyl modification of atleast the penultimate position from the 5′ terminus of the antisensestrand to reduce off-target effects; see, e.g., US 2007/0223427).

In one embodiment, the DsiRNA comprises the following (an exemplary“left-extended”, “DNA extended” DsiRNA):

5′-DNXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′3′-DNXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)XX-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. “Z”=DNA or RNA. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. “Z”=DNA or RNA. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)ZZXXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strandis the sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)ZZXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, 5 or 6. “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises:

5′-[X1/D1]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-[X2/D2]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1 to 50 or more, butis optionally 1-8 or 1-10, where at least one D1_(N) is present in thetop strand and is base paired with a corresponding D2_(N) in the bottomstrand. Optionally, D1_(N) and D1_(N+1) are base paired withcorresponding D2_(N) and D2_(N+1); D1_(N), D1_(N+1) and D1_(N+2) arebase paired with corresponding D2_(N), D1_(N+1) and D1_(N+2), etc.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In a related embodiment, the DsiRNA comprises:

5′-[X1/D1]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′3′-[X2/D2]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “D”=DNA, “Y” is an optional overhang domain comprisedof 0-10 RNA monomers that are optionally 2′-O-methyl RNA monomers—incertain embodiments, “Y” is an overhang domain comprised of 1-4 RNAmonomers that are optionally 2′-O-methyl RNA monomers, and “N”=1 to 50or more, but is optionally 1-8 or 1-10, where at least one D1_(N) ispresent in the top strand and is base paired with a corresponding D2_(N)in the bottom strand. Optionally, D1_(N) and D1_(N+1) are base pairedwith corresponding D2_(N) and D2_(N+1); D1_(N), D1_(N+1) and D1_(N+2)are base paired with corresponding D2_(N), D1_(N+1) and D1_(N+2), etc.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another). An exemplary DNA:DNA-extended DsiRNA agent with twoterminal mismatched residues is shown:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*) ^(M) ^(M) -3′3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*) _(M) _(M) -5′wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-8 or 1-10. “N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or6. Any of the residues of such agents can optionally be 2′-O-methyl RNAmonomers—alternating positioning of 2′-O-methyl RNA monomers thatcommences from the 3′-terminal residue of the bottom (second) strand, asshown for above asymmetric agents, can also be used in the above“blunt/fray” DsiRNA agent. In one embodiment, the top strand (firststrand) is the sense strand, and the bottom strand (second strand) isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. Modification andDNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In another embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsRNA structure. Anexemplary structure for such a molecule is shown:

5′-XXDDXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′ 3′-DDXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand. The above structure is modeled to forceDicer to cleave a minimum of a 21mer duplex as its primarypost-processing form. In embodiments where the bottom strand of theabove structure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand is likely to reduce off-targeteffects (as prior studies have shown a 2′-O-methyl modification of atleast the penultimate position from the 5′ terminus of the antisensestrand to reduce off-target effects; see, e.g., US 2007/0223427).

In certain embodiments, the “D” residues of the above structures includeat least one PS-DNA or PS-RNA. Optionally, the “D” residues of the abovestructures include at least one modified nucleotide that inhibits Dicercleavage.

While the above-described “DNA-extended” DsiRNA agents can becategorized as either “left extended” or “right extended”, DsiRNA agentscomprising both left- and right-extended DNA-containing sequences withina single agent (e.g., both flanks surrounding a core dsRNA structure aredsDNA extensions) can also be generated and used in similar manner tothose described herein for “right-extended” and “left-extended” agents.

In some embodiments, the DsiRNA of the instant invention furthercomprises a linking moiety or domain that joins the sense and antisensestrands of a DNA:DNA-extended DsiRNA agent. Optionally, such a linkingmoiety domain joins the 3′ end of the sense strand and the 5′ end of theantisense strand. The linking moiety may be a chemical (non-nucleotide)linker, such as an oligomethylenediol linker, oligoethylene glycollinker, or other art-recognized linker moiety. Alternatively, the linkercan be a nucleotide linker, optionally including an extended loop and/ortetraloop.

In one embodiment, the DsiRNA agent has an asymmetric structure, withthe sense strand having a 25-base pair length, and the antisense strandhaving a 27-base pair length with a 1-4 base 3′-overhang (e.g., a onebase 3′-overhang, a two base 3′-overhang, a three base 3′-overhang or afour base 3′-overhang). In another embodiment, this DsiRNA agent has anasymmetric structure further containing 2 deoxynucleotides at the 3′ endof the sense strand.

In another embodiment, the DsiRNA agent has an asymmetric structure,with the antisense strand having a 25-base pair length, and the sensestrand having a 27-base pair length with a 1-4 base 3′-overhang (e.g., aone base 3′-overhang, a two base 3′-overhang, a three base 3′-overhangor a four base 3′-overhang). In another embodiment, this DsiRNA agenthas an asymmetric structure further containing 2 deoxynucleotides at the3′ end of the antisense strand.

Exemplary KRAS targeting DsiRNA agents of the invention include thefollowing: KRAS-355 Target Site

  5′-CAGGUCAAGAGGAGUACAGUGCAau-3′ (SEQ ID NO: 5; DP1148P)3′-UCGUCCAGUUCUCCUCAUGUCACGUUA-5′ (SEQ ID NO: 14; DP1151G)5′-AGCAGGUCAAGAGGAGUACAGUGCAAU-3′ (SEQ ID NO: 6; DP1149P)3′-UCGUCCAGUUCUCCUCAUGUCACGUUA-5′ (SEQ ID NO: 14; DP1151G)5′-AGCAGGUCAAGAGGAGUACAGUGCA^(U) ^(A) -3′ (SEQ ID NO: 7; DP1150P)3′-UCGUCCAGUUCUCCUCAUGUCACGU_(U) _(A) -5' (SEQ ID NO: 14; DP1151G)KRAS-940 Target Site

  5′-UUAGCAUUUGUUUUAGCAUUACCta-3′ (SEQ ID NO: 8; DP1136P)3′-AUAAUCGUAAACAAAAUCGUAAUGGAU-5′ (SEQ ID NO: 43; DP1139G)5′-UAUUAGCAUUUGUUUUAGCAUUACCUA-3′ (SEQ ID NO: 9; DP1137P)3′-AUAAUCGUAAACAAAAUCGUAAUGGAU-5′ (SEQ ID NO: 43; DP1139G)5′-UAUUAGCAUUUGUUUUAGCAUUACC^(C) ^(C) -3' (SEQ ID NO: 10; DP1145P)3′-AUAAUCGUAAACAAAAUCGUAAUGG_(A) _(U) -5' (SEQ ID NO: 43; DP1139G)KRAS-355*Alternative (Polymorphic) Target Site

  5′-CAGGUCAUGAGGAGUACAGUGCAau-3′ (SEQ ID NO: 132)3′-UCGUCCAGUACUCCUCAUGUCACGUUA-5′ (SEQ ID NO: 138)5′-AGCAGGUCAUGAGGAGUACAGUGCAAU-3′ (SEQ ID NO: 187)3′-UCGUCCAGUACUCCUCAUGUCACGUUA-5′ (SEQ ID NO: 138)5′-AGCAGGUCAUGAGGAGUACAGUGCA^(U) ^(A) -3′ (SEQ ID NO: 188)3′-UCGUCCAGUACUCCUCAUGUCACGU_(U) _(A) -5′ (SEQ ID NO: 138)

Other exemplary KRAS targeting DsiRNA agents of the invention includethe following:

Lengthy table referenced here US08372816-20130212-T00001 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US08372816-20130212-T00002 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US08372816-20130212-T00003 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US08372816-20130212-T00004 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US08372816-20130212-T00005 Please refer tothe end of the specification for access instructions.Projected 21mer target sequences for each DsiRNA are shown in Table 7.

TABLE 7  DsiRNA Target Sequences (21 mers) KRAS-204 Target:5′-TAGTTGGAGCTGGTGGCGTAG-3′ (SEQ ID NO: 6843) KRAS-204* Target:5′-TAGTTGGAGCTGTTGGCGTAG-3′ (SEQ ID NO: 6844) KRAS-341 Target:5′-GATATTCTCGACACAGCAGGT-3′ (SEQ ID NO: 6845) KRAS-341* Target:5′-GATATTCTCGACACAGCAGGT-3′ (SEQ ID NO: 6846) KRAS-343 Target:5′-TATTCTCGACACAGCAGGTCA-3′ (SEQ ID NO: 6847) KRAS-343* Target:5′-TATTCTCGACACAGCAGGTCA-3′ (SEQ ID NO: 6848) KRAS-355 Target:5′-AGCAGGTCAAGAGGAGTACAG-3′ (SEQ ID NO: 6849) KRAS-355* Target:5′-AGCAGGTCATGAGGAGTACAG-3′ (SEQ ID NO: 6850) KRAS-361 Target:5′-TCAAGAGGAGTACAGTGCAAT-3′ (SEQ ID NO: 6851) KRAS-361* Target:5′-TCATGAGGAGTACAGTGCAAT-3′ (SEQ ID NO: 6852) KRAS-371 Target:5′-TACAGTGCAATGAGGGACCAG-3′ (SEQ ID NO: 6853) KRAS-401 Target:5′-ACTGGGGAGGGCTTTCTTTGT-3′ (SEQ ID NO: 6854) KRAS-404 Target:5′-GGGGAGGGCTTTCTTTGTGTA-3′ (SEQ ID NO: 6855) KRAS-406 Target:5′-GGAGGGCTTTCTTTGTGTATT-3′ (SEQ ID NO: 6856) KRAS-410 Target:5′-GGCTTTCTTTGTGTATTTGCC-3′ (SEQ ID NO: 6857) KRAS-415 Target:5′-TCTTTGTGTATTTGCCATAAA-3′ (SEQ ID NO: 6858) KRAS-416 Target:5′-CTTTGTGTATTTGCCATAAAT-3′ (SEQ ID NO: 6859) KRAS-417 Target:5′-TTTGTGTATTTGCCATAAATA-3′ (SEQ ID NO: 6860) KRAS-418 Target:5′-TTGTGTATTTGCCATAAATAA-3′ (SEQ ID NO: 6861) KRAS-419 Target:5′-TGTGTATTTGCCATAAATAAT-3′ (SEQ ID NO: 6862) KRAS-420 Target:5′-GTGTATTTGCCATAAATAATA-3′ (SEQ ID NO: 6863) KRAS-429 Target:5′-CCATAAATAATACTAAATCAT-3′ (SEQ ID NO: 6864) KRAS-434 Target:5′-AATAATACTAAATCATTTGAA-3′ (SEQ ID NO: 6865) KRAS-438 Target:5′-ATACTAAATCATTTGAAGATA-3′ (SEQ ID NO: 6866) KRAS-440 Target:5′-ACTAAATCATTTGAAGATATT-3′ (SEQ ID NO: 6867) KRAS-445 Target:5′-ATCATTTGAAGATATTCACCA-3′ (SEQ ID NO: 6868) KRAS-450 Target:5′-TTGAAGATATTCACCATTATA-3′ (SEQ ID NO: 6869) KRAS-452 Target:5′-GAAGATATTCACCATTATAGA-3′ (SEQ ID NO: 6870) KRAS-508 Target:5′-ACCTATGGTCCTAGTAGGAAA-3′ (SEQ ID NO: 6871) KRAS-534 Target:5′-GTGATTTGCCTTCTAGAACAG-3′ (SEQ ID NO: 6872) KRAS-760 Target:5′-TTGATGATGCCTTCTATACAT-3′ (SEQ ID NO: 6873) KRAS-776 Target:5′-TACATTAGTTCGAGAAATTCG-3′ (SEQ ID NO: 6874) KRAS-786 Target:5′-CGAGAAATTCGAAAACATAAA-3′ (SEQ ID NO: 6875) KRAS-795 Target:5′-CGAAAACATAAAGAAAAGATG-3′ (SEQ ID NO: 6876) KRAS-800 Target:5′-ACATAAAGAAAAGATGAGCAA-3′ (SEQ ID NO: 6877) KRAS-800* Target:5′-ACATAAAGAAAAGATGAGCAA-3′ (SEQ ID NO: 6878) KRAS-920 Target:5′-TTTTGTACATTACACTAAATT-3′ (SEQ ID NO: 6879) KRAS-935 Target:5′-TAAATTATTAGCATTTGTTTT-3′ (SEQ ID NO: 6880) KRAS-940 Target:5′-TATTAGCATTTGTTTTAGCAT-3′ (SEQ ID NO: 6881) KRAS-1032 Target:5′-GAAGTTTTTTTTTCCTCTAAG-3′ (SEQ ID NO: 6882) KRAS-1048 Target:5′-CTAAGTGCCAGTATTCCCAGA3′ (SEQ ID NO: 6883) KRAS-1188 Target:5′-TTGGTGTGAAACAAATTAATG-3′ (SEQ ID NO: 6884) KRAS-1256 Target:5′-GGATTAATTACTAATTTCAGT-3′ (SEQ ID NO: 6885) KRAS-1634 Target:5′-GCTATATTTACATGCTACTAA-3′ (SEQ ID NO: 6886) KRAS-5134 Target:5′-ATTTTAACTATTTTTGTATAG-3′ (SEQ ID NO: 6887) KRAS-5208 Target:5′-TGCAGTGTGATCCAGTTGTTT-3′ (SEQ ID NO: 6888) KRAS-174 Target:5′-TGCTGAAAATGACTGAATATA-3′ (SEQ ID NO: 6889) KRAS-180 Target:5′-AAATGACTGAATATAAACTTG-3′ (SEQ ID NO: 6890) KRAS-184 Target:5′-GACTGAATATAAACTTGTGGT-3′ (SEQ ID NO: 6891) KRAS-246 Target:5′-AGCTAATTCAGAATCATTTTG-3′ (SEQ ID NO: 6892) KRAS-270 Target:5′-ACGAATATGATCCAACAATAG-3′ (SEQ ID NO: 6893) KRAS-306 Target:5′-AGCAAGTAGTAATTGATGGAG-3′ (SEQ ID NO: 6894) KRAS-406 Target:5′-GGAGGGCTTTCTTTGTGTATT-3′ (SEQ ID NO: 6895) KRAS-415 Target:5′-TCTTTGTGTATTTGCCATAAA-3′ (SEQ ID NO: 6896) KRAS-416 Target:5′-CTTTGTGTATTTGCCATAAAT-3′ (SEQ ID NO: 6897) KRAS-417 Target:5′-TTTGTGTATTTGCCATAAATA-3′ (SEQ ID NO: 6898) KRAS-418 Target:5′-TTGTGTATTTGCCATAAATAA-3′ (SEQ ID NO: 6899) KRAS-422 Target:5′-GTATTTGCCATAAATAATACT-3′ (SEQ ID NO: 6900) KRAS-423 Target:5′-TATTTGCCATAAATAATACTA73′ (SEQ ID NO: 6901) KRAS-429 Target:5′-CCATAAATAATACTAAATCAT-3′ (SEQ ID NO: 6902) KRAS-430 Target:5′-CATAAATAATACTAAATCATT-3′ (SEQ ID NO: 6903) KRAS-432 Target:5′-TAAATAATACTAAATCATTTG-3′ (SEQ ID NO: 6904) KRAS-433 Target:5′-AAATAATACTAAATCATTTGA-3′ (SEQ ID NO: 6905) KRAS-434 Target:5′-AATAATACTAAATCATTTGAA-3′ (SEQ ID NO: 6906) KRAS-435 Target:5′-ATAATACTAAATCATTTGAAG-3′ (SEQ ID NO: 6907) KRAS-448 Target:5′-ATTTGAAGATATTCACCATTA-3′ (SEQ ID NO: 6908) KRAS-450 Target:5′-TTGAAGATATTCACCATTATA-3′ (SEQ ID NO: 6909) KRAS-451 Target:5′-TGAAGATATTCACCATTATAG-3′ (SEQ ID NO: 6910) KRAS-452 Target:5′-GAAGATATTCACCATTATAGA-3′ (SEQ ID NO: 6911) KRAS-453 Target:5′-AAGATATTCACCATTATAGAG-3′ (SEQ ID NO: 6912) KRAS-463 Target:5′-CCATTATAGAGAACAAATTAA-3′ (SEQ ID NO: 6913) KRAS-465 Target:5′-ATTATAGAGAACAAATTAAAA-3′ (SEQ ID NO: 6914) KRAS-466 Target:5′-TTATAGAGAACAAATTAAAAG-3′ (SEQ ID NO: 6915) KRAS-467 Target:5′-TATAGAGAACAAATTAAAAGA-3′ (SEQ ID NO: 6916) KRAS-468 Target:5′-ATAGAGAACAAATTAAAAGAG-3′ (SEQ ID NO: 6917) KRAS-518 Target:5′-CTAGTAGGAAATAAATGTGAT-3′ (SEQ ID NO: 6918) KRAS-519 Target:5′-TAGTAGGAAATAAATGTGATT-3′ (SEQ ID NO: 6919) KRAS-583 Target:5′-AAGAAGTTATGGAATTCCTTT-3′ (SEQ ID NO: 6920) KRAS-586 Target:5′-AAGTTATGGAATTCCTTTTAT-3′ (SEQ ID NO: 6921) KRAS-587 Target:5′-AGTTATGGAATTCCTTTTATT-3′ (SEQ ID NO: 6922) KRAS-588 Target:5′-GTTATGGAATTCCTTTTATTG-3′ (SEQ ID NO: 6923) KRAS-589 Target:5′-TTATGGAATTCCTTTTATTGA-3′ (SEQ ID NO: 6924) KRAS-636 Target:5′-TGGAGGATGCTTTTTATACAT-3′ (SEQ ID NO: 6925) KRAS-638 Target:5′-GAGGATGCTTTTTATACATTG-3′ (SEQ ID NO: 6926) KRAS-678 Target:5′-ACAGATTGAAAAAAATCAGCA-3′ (SEQ ID NO: 6927) KRAS-679 Target:5′-CAGATTGAAAAAAATCAGCAA-3′ (SEQ ID NO: 6928) KRAS-681 Target:5′-GATTGAAAAAAATCAGCAAAG-3′ (SEQ ID NO: 6929) KRAS-682 Target:5′-ATTGAAAAAAATCAGCAAAGA-3′ (SEQ ID NO: 6930) KRAS-683 Target:5′-TTGAAAAAAATCAGCAAAGAA-3′ (SEQ ID NO: 6931) KRAS-684 Target:5′-TGAAAAAAATCAGCAAAGAAG-3′ (SEQ ID NO: 6932) KRAS-712 Target:5′-TCCTGGCTGTGTGAAAATTAA-3′ (SEQ ID NO: 6933) KRAS-716 Target:5′-GGCTGTGTGAAAATTAAAAAA-3′ (SEQ ID NO: 6934) KRAS-720 Target:5′-GTGTGAAAATTAAAAAATGCA-3′ (SEQ ID NO: 6935) KRAS-721 Target:5′-TGTGAAAATTAAAAAATGCAT-3′ (SEQ ID NO: 6936) KRAS-722 Target:5′-GTGAAAATTAAAAAATGCATT-3′ (SEQ ID NO: 6937) KRAS-723 Target:5′-TGAAAATTAAAAAATGCATTA-3′ (SEQ ID NO: 6938) KRAS-725 Target:5′-AAAATTAAAAAATGCATTATA-3′ (SEQ ID NO: 6939) KRAS-726 Target:5′-AAATTAAAAAATGCATTATAA-3′ (SEQ ID NO: 6940) KRAS-727 Target:5′-AATTAAAAAATGCATTATAAT-3′ (SEQ ID NO: 6941) KRAS-728 Target:5′-ATTAAAAAATGCATTATAATG-3′ (SEQ ID NO: 6942) KRAS-730 Target:5′-TAAAAAATGCATTATAATGTA-3′ (SEQ ID NO: 6943) KRAS-737 Target:5′-TGCATTATAATGTAATCTGGG-3′ (SEQ ID NO: 6944) KRAS-784 Target:5′-TTCGAGAAATTCGAAAACATA-3′ (SEQ ID NO: 6945) KRAS-785 Target:5′-TCGAGAAATTCGAAAACATAA-3′ (SEQ ID NO: 6946) KRAS-786 Target:5′-CGAGAAATTCGAAAACATAAA-3′ (SEQ ID NO: 6947) KRAS-789 Target:5′-GAAATTCGAAAACATAAAGAA-3′ (SEQ ID NO: 6948) KRAS-794 Target:5′-TCGAAAACATAAAGAAAAGAT-3′ (SEQ ID NO: 6949) KRAS-795 Target:5′-CGAAAACATAAAGAAAAGATG-3′ (SEQ ID NO: 6950) KRAS-819 Target:5′-AAAGATGGTAAAAAGAAGAAA-3′ (SEQ ID NO: 6951) KRAS-820 Target:5′-AAGATGGTAAAAAGAAGAAAA-3′ (SEQ ID NO: 6952) KRAS-822 Target:5′-GATGGTAAAAAGAAGAAAAAG-3′ (SEQ ID NO: 6953) KRAS-823 Target:5′-ATGGTAAAAAGAAGAAAAAGA-3′ (SEQ ID NO: 6954) KRAS-828 Target:5′-AAAAAGAAGAAAAAGAAGTCA-3′ (SEQ ID NO: 6955) KRAS-829 Target:5′-AAAAGAAGAAAAAGAAGTCAA-3′ (SEQ ID NO: 6956) KRAS-830 Target:5′-AAAGAAGAAAAAGAAGTCAAA-3′ (SEQ ID NO: 6957) KRAS-846 Target:5′-TCAAAGACAAAGTGTGTAATT-3′ (SEQ ID NO: 6958) KRAS-847 Target:5′-CAAAGACAAAGTGTGTAATTA-3′ (SEQ ID NO: 6959) KRAS-851 Target:5′-GACAAAGTGTGTAATTATGTA-3′ (SEQ ID NO: 6960) KRAS-853 Target:5′-CAAAGTGTGTAATTATGTAAA-3′ (SEQ ID NO: 6961) KRAS-857 Target:5′-GTGTGTAATTATGTAAATACA-3′ (SEQ ID NO: 6962) KRAS-859 Target:5′-GTGTAATTATGTAAATACAAT-3′ (SEQ ID NO: 6963) KRAS-861 Target:5′-GTAATTATGTAAATACAATTT-3′ (SEQ ID NO: 6964) KRAS-862 Target:5′-TAATTATGTAAATACAATTTG-3′ (SEQ ID NO: 6965) KRAS-866 Target:5′-TATGTAAATACAATTTGTACT-3′ (SEQ ID NO: 6966) KRAS-867 Target:5′-ATGTAAATACAATTTGTACTT-3′ (SEQ ID NO: 6967) KRAS-868 Target:5′-TGTAAATACAATTTGTACTTT-3′ (SEQ ID NO: 6968) KRAS-869 Target:5′-GTAAATACAATTTGTACTTTT-3′ (SEQ ID NO: 6969) KRAS-875 Target:5′-ACAATTTGTACTTTTTTCTTA-3′ (SEQ ID NO: 6970) KRAS-876 Target:5′-CAATTTGTACTTTTTTCTTAA-3′ (SEQ ID NO: 6971) KRAS-901 Target:5′-TACTAGTACAAGTGGTAATTT-3′ (SEQ ID NO: 6972) KRAS-902 Target:5′-ACTAGTACAAGTGGTAATTTT-3′ (SEQ ID NO: 6973) KRAS-908 Target:5′-ACAAGTGGTAATTTTTGTACA-3′ (SEQ ID NO: 6974) KRAS-909 Target:5′-CAAGTGGTAATTTTTGTACAT-3′ (SEQ ID NO: 6975) KRAS-916 Target:5′-TAATTTTTGTACATTACACTA-3′ (SEQ ID NO: 6976) KRAS-917 Target:5′-AATTTTTGTACATTACACTAA-3′ (SEQ ID NO: 6977) KRAS-918 Target:5′-ATTTTTGTACATTACACTAAA-3′ (SEQ ID NO: 6978) KRAS-919 Target:5′-TTTTTGTACATTACACTAAAT-3′ (SEQ ID NO: 6979) KRAS-920 Target:5′-TTTTGTACATTACACTAAATT-3′ (SEQ ID NO: 6980) KRAS-932 Target:5′-CACTAAATTATTAGCATTTGT-3′ (SEQ ID NO: 6981) KRAS-934 Target:5′-CTAAATTATTAGCATTTGTTT-3′ (SEQ ID NO: 6982) KRAS-935 Target:5′-TAAATTATTAGCATTTGTTTT-3′ (SEQ ID NO: 6983) KRAS-940 Target:5′-TATTAGCATTTGTTTTAGCAT-3′ (SEQ ID NO: 6984) KRAS-946 Target:5′-CATTTGTTTTAGCATTACCTA-3′ (SEQ ID NO: 6985) KRAS-947 Target:5′-ATTTGTTTTAGCATTACCTAA-3′ (SEQ ID NO: 6986) KRAS-949 Target:5′-TTGTTTTAGCATTACCTAATT-3′ (SEQ ID NO: 6987) KRAS-950 Target:5′-TGTTTTAGCATTACCTAATTT-3′ (SEQ ID NO: 6988) KRAS-951 Target:5′-GTTTTAGCATTACCTAATTTT-3′ (SEQ ID NO: 6989) KRAS-952 Target:5′-TTTTAGCATTACCTAATTTTT-3′ (SEQ ID NO: 6990) KRAS-988 Target:5′-GACTGTTAGCTTTTACCTTAA-3′ (SEQ ID NO: 6991) KRAS-989 Target:5′-ACTGTTAGCTTTTACCTTAAA-3′ (SEQ ID NO: 6992) KRAS-995 Target:5′-AGCTTTTACCTTAAATGCTTA-3′ (SEQ ID NO: 6993) KRAS-996 Target:5′-GCTTTTACCTTAAATGCTTAT-3′ (SEQ ID NO: 6994) KRAS-997 Target:5′-CTTTTACCTTAAATGCTTATT-3′ (SEQ ID NO: 6995) KRAS-1003 Target:5′-CCTTAAATGCTTATTTTAAAA-3′ (SEQ ID NO: 6996) KRAS-1004 Target:5′-CTTAAATGCTTATTTTAAAAT-3′ (SEQ ID NO: 6997) KRAS-1010 Target:5′-TGCTTATTTTAAAATGACAGT-3′ (SEQ ID NO: 6998) KRAS-1012 Target:5′-CTTATTTTAAAATGACAGTGG-3′ (SEQ ID NO: 6999) KRAS-1029 Target:5′-GTGGAAGTTTTTTTTTCCTCT-3′ (SEQ ID NO: 7000) KRAS-1031 Target:5′-GGAAGTTTTTTTTTCCTCTAA-3′ (SEQ ID NO: 7001) KRAS-1032 Target:5′-GAAGTTTTTTTTTCCTCTAAG-3′ (SEQ ID NO: 7002) KRAS-1070 Target:5′-TTTTGGTTTTTGAACTAGCAA-3′ (SEQ ID NO: 7003) KRAS-1092 Target:5′-GCCTGTGAAAAAGAAACTGAA-3′ (SEQ ID NO: 7004) KRAS-1099 Target:5′-AAAAAGAAACTGAATACCTAA-3′ (SEQ ID NO: 7005) KRAS-1100 Target:5′-AAAAGAAACTGAATACCTAAG-3′ (SEQ ID NO: 7006) KRAS-1106 Target:5′-AACTGAATACCTAAGATTTCT-3′ (SEQ ID NO: 7007) KRAS-1147 Target:5′-TGCAGTTGATTACTTCTTATT-3′ (SEQ ID NO: 7008) KRAS-1148 Target:5′-GCAGTTGATTACTTCTTATTT-3′ (SEQ ID NO: 7009) KRAS-1152 Target:5′-TTGATTACTTCTTATTTTTCT-3′ (SEQ ID NO: 7010) KRAS-1160 Target:5′-TTCTTATTTTTCTTACCAATT-3′ (SEQ ID NO: 7011) KRAS-1161 Target:5′-TCTTATTTTTCTTACCAATTG-3′ (SEQ ID NO: 7012) KRAS-1162 Target:5′-CTTATTTTTCTTACCAATTGT-3′ (SEQ ID NO: 7013) KRAS-1185 Target:5′-ATGTTGGTGTGAAACAAATTA-3′ (SEQ ID NO: 7014) KRAS-1188 Target:5′-TTGGTGTGAAACAAATTAATG-3′ (SEQ ID NO: 7015) KRAS-1189 Target:5′-TGGTGTGAAACAAATTAATGA-3′ (SEQ ID NO: 7016) KRAS-1197 Target:5′-AACAAATTAATGAAGCTTTTG-3′ (SEQ ID NO: 7017) KRAS-1198 Target:5′-ACAAATTAATGAAGCTTTTGA-3′ (SEQ ID NO: 7018) KRAS-1230 Target:5′-TCTGTGTTTTATCTAGTCACA-3′ (SEQ ID NO: 7019) KRAS-1231 Target:5′-CTGTGTTTTATCTAGTCACAT-3′ (SEQ ID NO: 7020) KRAS-1234 Target:5′-TGTTTTATCTAGTCACATAAA-3′ (SEQ ID NO: 7021) KRAS-1235 Target:5′-GTTTTATCTAGTCACATAAAT-3′ (SEQ ID NO: 7022) KRAS-1240 Target:5′-ATCTAGTCACATAAATGGATT-3′ (SEQ ID NO: 7023) KRAS-1241 Target:5′-TCTAGTCACATAAATGGATTA-3′ (SEQ ID NO: 7024) KRAS-1249 Target:5′-CATAAATGGATTAATTACTAA-3′ (SEQ ID NO: 7025) KRAS-1250 Target:5′-ATAAATGGATTAATTACTAAT-3′ (SEQ ID NO: 7026) KRAS-1255 Target:5′-TGGATTAATTACTAATTTCAG-3′ (SEQ ID NO: 7027) KRAS-1257 Target:5′-GATTAATTACTAATTTCAGTT-3′ (SEQ ID NO: 7028) KRAS-1258 Target:5′-ATTAATTACTAATTTCAGTTG-3′ (SEQ ID NO: 7029) KRAS-1259 Target:5′-TTAATTACTAATTTCAGTTGA-3′ (SEQ ID NO: 7030) KRAS-1287 Target:5′-TAATTGGTTTTTACTGAAACA-3′ (SEQ ID NO: 7031) KRAS-1291 Target:5′-TGGTTTTTACTGAAACATTGA-3′ (SEQ ID NO: 7032) KRAS-1292 Target:5′-GGTTTTTACTGAAACATTGAG-3′ (SEQ ID NO: 7033) KRAS-1307 Target:5′-ATTGAGGGAACACAAATTTAT-3′ (SEQ ID NO: 7034) KRAS-1308 Target:5′-TTGAGGGAACACAAATTTATG-3′ (SEQ ID NO: 7035) KRAS-1381 Target:5′-TGATGAATGTAAAGTTACACT-3′ (SEQ ID NO: 7036) KRAS-1382 Target:5′-GATGAATGTAAAGTTACACTG-3′ (SEQ ID NO: 7037) KRAS-1454 Target:5′-CCAAAATATTATATTTTTTCT-3′ (SEQ ID NO: 7038) KRAS-1455 Target:5′-CAAAATATTATATTTTTTCTA-3′ (SEQ ID NO: 7039) KRAS-1456 Target:5′-AAAAtATTATATTTTTTCTAT-3′ (SEQ ID NO: 7040) KRAS-1457 Target:5′-AAATATTATATTTTTTCTATA-3′ (SEQ ID NO: 7041) KRAS-1459 Target:5′-ATATTATATTTTTTCTATAAA-3′ (SEQ ID NO: 7042) KRAS-1460 Target:5′-TATTATATTTTTTCTATAAAA-3′ (SEQ ID NO: 7043) KRAS-1461 Target:5′-ATTATATTTTTTCTATAAAAA-3′ (SEQ ID NO: 7044) KRAS-1462 Target:5′-TTATATTTTTTCTATAAAAAG-3′ (SEQ ID NO: 7045) KRAS-1463 Target:5′-TATATTTTTTCTATAAAAAGA-3′ (SEQ ID NO: 7046) KRAS-1464 Target:5′-ATATTTTTTCTATAAAAAGAA-3′ (SEQ ID NO: 7047) KRAS-1465 Target:5′-TATTTTTTCTATAAAAAGAAA-3′ (SEQ ID NO: 7048) KRAS-1466 Target:5′-ATTTTTTCTATAAAAAGAAAA-3′ (SEQ ID NO: 7049) KRAS-1471 Target:5′-TTCTATAAAAAGAAAAAAATG-3′ (SEQ ID NO: 7050) KRAS-1472 Target:5′-TCTATAAAAAGAAAAAAATGG-3′ (SEQ ID NO: 7051) KRAS-1474 Target:5′-TATAAAAAGAAAAAAATGGAA-3′ (SEQ ID NO: 7052) KRAS-1475 Target:5′-ATAAAAAGAAAAAAATGGAAA-3′ (SEQ ID NO: 7053) KRAS-1476 Target:5′-TAAAAAGAAAAAAATGGAAAA-3′ (SEQ ID NO: 7054) KRAS-1477 Target:5′-AAAAAGAAAAAAATGGAAAAA-3′ (SEQ ID NO: 7055) KRAS-1478 Target:5′-AAAAGAAAAAAATGGAAAAAA-3′ (SEQ ID NO: 7056) KRAS-1479 Target:5′-AAAGAAAAAAATGGAAAAAAA-3′ (SEQ ID NO: 7057) KRAS-1480 Target:5′-AAGAAAAAAATGGAAAAAAAT-3′ (SEQ ID NO: 7058) KRAS-1484 Target:5′-AAAAAATGGAAAAAAATTACA-3′ (SEQ ID NO: 7059) KRAS-1485 Target:5′-AAAAATGGAAAAAAATTACAA-3′ (SEQ ID NO: 7060) KRAS-1490 Target:5′-TGGAAAAAAATTACAAGGCAA-3′ (SEQ ID NO: 7061) KRAS-1491 Target:5′-GGAAAAAAATTACAAGGCAAT-3′ (SEQ ID NO: 7062) KRAS-1492 Target:5′-GAAAAAAATTACAAGGCAATG-3′ (SEQ ID NO: 7063) KRAS-1527 Target:5′-GCCATTTCCTTTTCACATTAG-3′ (SEQ ID NO: 7064) KRAS-1533 Target:5′-TCCTTTTCACATTAGATAAAT-3′ (SEQ ID NO: 7065) KRAS-1540 Target:5′-CACATTAGATAAATTACTATA-3′ (SEQ ID NO: 7066) KRAS-1541 Target:5′-ACATTAGATAAATTACTATAA-3′ (SEQ ID NO: 7067) KRAS-1542 Target:5′-CATTAGATAAATTACTATAAA-3′ (SEQ ID NO: 7068) KRAS-1597 Target:5′-AGTATGAAATGGGGATTATTA-3′ (SEQ ID NO: 7069) KRAS-1606 Target:5′-TGGGGATTATTATAGCAACCA-3′ (SEQ ID NO: 7070) KRAS-1633 Target:5′-GGCTATATTTACATGCTACTA-3′ (SEQ ID NO: 7071) KRAS-1634 Target:5′-GCTATATTTACATGCTACTAA-3′ (SEQ ID NO: 7072) KRAS-1635 Target:5′-CTATATTTACATGCTACTAAA-3′ (SEQ ID NO: 7073) KRAS-1636 Target:5′-TATATTTACATGCTACTAAAT-3′ (SEQ ID NO: 7074) KRAS-1637 Target:5′-ATATTTACATGCTACTAAATT-3′ (SEQ ID NO: 7075) KRAS-1642 Target:5′-TACATGCTACTAAATTTTTAT-3′ (SEQ ID NO: 7076) KRAS-1649 Target:5′-TACTAAATTTTTATAATAATT-3′ (SEQ ID NO: 7077) KRAS-1650 Target:5′-ACTAAATTTTTATAATAATTG-3′ (SEQ ID NO: 7078) KRAS-1652 Target:5′-TAAATTTTTATAATAATTGAA-3′ (SEQ ID NO: 7079) KRAS-1653 Target:5′-AAATTTTTATAATAATTGAAA-3′ (SEQ ID NO: 7080) KRAS-1654 Target:5′-AATTTTTATAATAATTGAAAA-3′ (SEQ ID NO: 7081) KRAS-1655 Target:5′-ATTTTTATAATAATTGAAAAG-3′ (SEQ ID NO: 7082) KRAS-1656 Target:5′-TTTTTATAATAATTGAAAAGA-3′ (SEQ ID NO: 7083) KRAS-1657 Target:5′-TTTTATAATAATTGAAAAGAT-3′ (SEQ ID NO: 7084) KRAS-1658 Target:5′-TTTATAATAATTGAAAAGATT-3′ (SEQ ID NO: 7085) KRAS-1659 Target:5′-TTATAATAATTGAAAAGATTT-3′ (SEQ ID NO: 7086) KRAS-1660 Target:5′-TATAATAATTGAAAAGATTTT-3′ (SEQ ID NO: 7087) KRAS-1664 Target:5′-ATAATTGAAAAGATTTTAACA-3′ (SEQ ID NO: 7088) KRAS-1665 Target:5′-TAATTGAAAAGATTTTAACAA-3′ (SEQ ID NO: 7089) KRAS-1667 Target:5′-ATTGAAAAGATTTTAACAAGT-3′ (SEQ ID NO: 7090) KRAS-1668 Target:5′-TTGAAAAGATTTTAACAAGTA-3′ (SEQ ID NO: 7091) KRAS-1669 Target:5′-TGAAAAGATTTTAACAAGTAT-3′ (SEQ ID NO: 7092) KRAS-1670 Target:5′-GAAAAGATTTTAACAAGTATA-3′ (SEQ ID NO: 7093) KRAS-1671 Target:5′-AAAAGATTTTAACAAGTATAA-3′ (SEQ ID NO: 7094) KRAS-1672 Target:5′-AAAGATTTTAACAAGTATAAA-3′ (SEQ ID NO: 7095) KRAS-1673 Target:5′-AAGATTTTAACAAGTATAAAA-3′ (SEQ ID NO: 7096) KRAS-1674 Target:5′-AGATTTTAACAAGTATAAAAA-3′ (SEQ ID NO: 7097) KRAS-1675 Target:5′-GATTTTAACAAGTATAAAAAA-3′ (SEQ ID NO: 7098) KRAS-1682 Target:5′-ACAAGTATAAAAAATTCTCAT-3′ (SEQ ID NO: 7099) KRAS-1683 Target:5′-CAAGTATAAAAAATTCTCATA-3′ (SEQ ID NO: 7100) KRAS-1684 Target:5′-AAGTATAAAAAATTCTCATAG-3′ (SEQ ID NO: 7101) KRAS-1685 Target:5′-AGTATAAAAAATTCTCATAGG-3′ (SEQ ID NO: 7102) KRAS-1686 Target:5′-GTATAAAAAATTCTCATAGGA-3′ (SEQ ID NO: 7103) KRAS-1687 Target:5′-TATAAAAAATTCTCATAGGAA-3′ (SEQ ID NO: 7104) KRAS-1688 Target:5′-ATAAAAAATTCTCATAGGAAT-3′ (SEQ ID NO: 7105) KRAS-1689 Target:5′-TAAAAAATTCTCATAGGAATT-3′ (SEQ ID NO: 7106) KRAS-1691 Target:5′-AAAAATTCTCATAGGAATTAA-3′ (SEQ ID NO: 7107) KRAS-1692 Target:5′-AAAATTCTCATAGGAATTAAA-3′ (SEQ ID NO: 7108) KRAS-1736 Target:5′-CTCTTTCATAGTATAACTTTA-3′ (SEQ ID NO: 7109) KRAS-1741 Target:5′-TCATAGTATAACTTTAAATCT-3′ (SEQ ID NO: 7110) KRAS-1742 Target:5′-CATAGTATAACTTTAAATCTT-3′ (SEQ ID NO: 7111) KRAS-1753 Target:5′-TTTAAATCTTTTCTTCAACTT-3′ (SEQ ID NO: 7112) KRAS-1754 Target:5′-TTAAATCTTTTCTTCAACTTG-3′ (SEQ ID NO: 7113) KRAS-1769 Target:5′-AACTTGAGTCTTTGAAGATAG-3′ (SEQ ID NO: 7114) KRAS-1771 Target:5′-CTTGAGTCTTTGAAGATAGTT-3′ (SEQ ID NO: 7115) KRAS-1772 Target:5′-TTGAGTCTTTGAAGATAGTTT-3′ (SEQ ID NO: 7116) KRAS-1783 Target:5′-AAGATAGTTTTAATTCTGCTT-3′ (SEQ ID NO: 7117) KRAS-1784 Target:5′-AGATAGTTTTAATTCTGCTTG-3′ (SEQ ID NO: 7118) KRAS-1785 Target:5′-GATAGTTTTAATTCTGCTTGT-3′ (SEQ ID NO: 7119) KRAS-1799 Target:5′-TGCTTGTGACATTAAAAGATT-3′ (SEQ ID NO: 7120) KRAS-2047 Target:5′-AGCATTGCTTTTGTTTCTTAA-3′ (SEQ ID NO: 7121) KRAS-2048 Target:5′-GCATTGCTTTTGTTTCTTAAG-3′ (SEQ ID NO: 7122) KRAS-2054 Target:5′-CTTTTGTTTCTTAAGAAAACA-3′ (SEQ ID NO: 7123) KRAS-2061 Target:5′-TTCTTAAGAAAACAAACTCTT-3′ (SEQ ID NO: 7124) KRAS-2062 Target:5′-TCTTAAGAAAACAAACTCTTT-3′ (SEQ ID NO: 7125) KRAS-2063 Target:5′-CTTAAGAAAACAAACTCTTTT-3′ (SEQ ID NO: 7126) KRAS-2064 Target:5′-TTAAGAAAACAAACTCTTTTT-3′ (SEQ ID NO: 7127) KRAS-2065 Target:5′-TAAGAAAACAAACTCTTTTTT-3′ (SEQ ID NO: 7128) KRAS-2066 Target:5′-AAGAAAACAAACTCTTTTTTA-3′ (SEQ ID NO: 7129) KRAS-2067 Target:5′-AGAAAACAAACTCTTTTTTAA-3′ (SEQ ID NO: 7130) KRAS-2071 Target:5′-AACAAACTCTTTTTTAAAAAT-3′ (SEQ ID NO: 7131) KRAS-2077 Target:5′-CTCTTTTTTAAAAATTACTTT-3′ (SEQ ID NO: 7132) KRAS-2078 Target:5′-TCTTTTTTAAAAATTACTTTT-3′ (SEQ ID NO: 7133) KRAS-2079 Target:5′-CTTTTTTAAAAATTACTTTTA-3′ (SEQ ID NO: 7134) KRAS-2080 Target:5′-TTTTTTAAAAATTACTTTTAA-3′ (SEQ ID NO: 7135) KRAS-2081 Target:5′-TTTTTAAAAATTACTTTTAAA-3′ (SEQ ID NO: 7136) KRAS-2082 Target:5′-TTTTAAAAATTACTTTTAAAT-3′ (SEQ ID NO: 7137) KRAS-2083 Target:5′-TTTAAAAATTACTTTTAAATA-3′ (SEQ ID NO: 7138) KRAS-2084 Target:5′-TTAAAAATTACTTTTAAATAT-3′ (SEQ ID NO: 7139) KRAS-2085 Target:5′-TAAAAATTACTTTTAAATATT-3′ (SEQ ID NO: 7140) KRAS-2092 Target:5′-TACTTTTAAATATTAACTCAA-3′ (SEQ ID NO: 7141) KRAS-2093 Target:5′-ACTTTTAAATATTAACTCAAA-3′ (SEQ ID NO: 7142) KRAS-2095 Target:5′-TTTTAAATATTAACTCAAAAG-3′ (SEQ ID NO: 7143) KRAS-2097 Target:5′-TTAAATATTAACTCAAAAGTT-3′ (SEQ ID NO: 7144) KRAS-2098 Target:5′-TAAATATTAACTCAAAAGTTG-3′ (SEQ ID NO: 7145) KRAS-2099 Target:5′-AAATATTAACTCAAAAGTTGA-3′ (SEQ ID NO: 7146) KRAS-2100 Target:5′-AATATTAACTCAAAAGTTGAG-3′ (SEQ ID NO: 7147) KRAS-2134 Target:5′-GGTGTGCCAAGACATTAATTT-3′ (SEQ ID NO: 7148) KRAS-2139 Target:5′-GCCAAGACATTAATTTTTTTT-3′ (SEQ ID NO: 7149) KRAS-2140 Target:5′-CCAAGACATTAATTTTTTTTT-3′ (SEQ ID NO: 7150) KRAS-2146 Target:5′-CATTAATTTTTTTTTTAAACA-3′ (SEQ ID NO: 7151) KRAS-2147 Target:5′-ATTAATTTTTTTTTTAAACAA-3′ (SEQ ID NO: 7152) KRAS-2149 Target:5′-TAATTTTTTTTTTAAACAATG-3′ (SEQ ID NO: 7153) KRAS-2151 Target:5′-ATTTTTTTTTTAAACAATGAA-3′ (SEQ ID NO: 7154) KRAS-2152 Target:5′-TTTTTTTTTTAAACAATGAAG-3′ (SEQ ID NO: 7155) KRAS-2153 Target:5′-TTTTTTTTTAAACAATGAAGT-3′ (SEQ ID NO: 7156) KRAS-2154 Target:5′-TTTTTTTTAAACAATGAAGTG-3′ (SEQ ID NO: 7157) KRAS-2155 Target:5′-TTTTTTTAAACAATGAAGTGA-3′ (SEQ ID NO: 7158) KRAS-2157 Target:5′-TTTTTAAACAATGAAGTGAAA-3′ (SEQ ID NO: 7159) KRAS-2158 Target:5′-TTTTAAACAATGAAGTGAAAA-3′ (SEQ ID NO: 7160) KRAS-2159 Target:5′-TTTAAACAATGAAGTGAAAAA-3′ (SEQ ID NO: 7161) KRAS-2167 Target:5′-ATGAAGTGAAAAAGTTTTACA-3′ (SEQ ID NO: 7162) KRAS-2173 Target:5′-TGAAAAAGTTTTACAATCTCT-3′ (SEQ ID NO: 7163) KRAS-2174 Target:5′-GAAAAAGTTTTACAATCTCTA-3′ (SEQ ID NO: 7164) KRAS-2175 Target:5′-AAAAAGTTTTACAATCTCTAG-3′ (SEQ ID NO: 7165) KRAS-2216 Target:5′-ACTGGTTAAATTAACATTGCA-3′ (SEQ ID NO: 7166) KRAS-2217 Target:5′-CTGGTTAAATTAACATTGCAT-3′ (SEQ ID NO: 7167) KRAS-2218 Target:5′-TGGTTAAATTAACATTGCATA-3′ (SEQ ID NO: 7168) KRAS-2229 Target:5′-ACATTGCATAAACACTTTTCA-3′ (SEQ ID NO: 7169) KRAS-2247 Target:5′-TCAAGTCTGATCCATATTTAA-3′ (SEQ ID NO: 7170) KRAS-2257 Target:5′-TCCATATTTAATAATGCTTTA-3′ (SEQ ID NO: 7171) KRAS-2258 Target:5′-CCATATTTAATAATGCTTTAA-3′ (SEQ ID NO: 7172) KRAS-2259 Target:5′-CATATTTAATAATGCTTTAAA-3′ (SEQ ID NO: 7173) KRAS-2260 Target:5′-ATATTTAATAATGCTTTAAAA-3′ (SEQ ID NO: 7174) KRAS-2261 Target:5′-TATTTAATAATGCTTTAAAAT-3′ (SEQ ID NO: 7175) KRAS-2262 Target:5′-ATTTAATAATGCTTTAAAATA-3′ (SEQ ID NO: 7176) KRAS-2264 Target:5′-TTAATAATGCTTTAAAATAAA-3′ (SEQ ID NO: 7177) KRAS-2265 Target:5′-TAATAATGCTTTAAAATAAAA-3′ (SEQ ID NO: 7178) KRAS-2266 Target:5′-AATAATGCTTTAAAATAAAAA-3′ (SEQ ID NO: 7179) KRAS-2267 Target:5′-ATAATGCTTTAAAATAAAAAT-3′ (SEQ ID NO: 7180) KRAS-2274 Target:5′-TTTAAAATAAAAATAAAAACA-3′ (SEQ ID NO: 7181) KRAS-2280 Target:5′-ATAAAAATAAAAACAATCCTT-3′ (SEQ ID NO: 7182) KRAS-2281 Target:5′-TAAAAATAAAAACAATCCTTT-3′ (SEQ ID NO: 7183) KRAS-2282 Target:5′-AAAAATAAAAACAATCCTTTT-3′ (SEQ ID NO: 7184) KRAS-2283 Target:5′-AAAATAAAAACAATCCTTTTG-3′ (SEQ ID NO: 7185) KRAS-2284 Target:5′-AAATAAAAACAATCCTTTTGA-3′ (SEQ ID NO: 7186) KRAS-2285 Target:5′-AATAAAAACAATCCTTTTGAT-3′ (SEQ ID NO: 7187) KRAS-2286 Target:5′-ATAAAAACAATCCTTTTGATA-3′ (SEQ ID NO: 7188) KRAS-2287 Target:5′-TAAAAACAATCCTTTTGATAA-3′ (SEQ ID NO: 7189) KRAS-2291 Target:5′-AACAATCCTTTTGATAAATTT-3′ (SEQ ID NO: 7190) KRAS-2296 Target:5′-TCCTTTTGATAAATTTAAAAT-3′ (SEQ ID NO: 7191) KRAS-2297 Target:5′-CCTTTTGATAAATTTAAAATG-3′ (SEQ ID NO: 7192) KRAS-2298 Target:5′-CTTTTGATAAATTTAAAATGT-3′ (SEQ ID NO: 7193) KRAS-2302 Target:5′-TGATAAATTTAAAATGTTACT-3′ (SEQ ID NO: 7194) KRAS-2303 Target:5′-GATAAATTTAAAATGTTACTT-3′ (SEQ ID NO: 7195) KRAS-2304 Target:5′-ATAAATTTAAAATGTTACTTA-3′ (SEQ ID NO: 7196) KRAS-2305 Target:5′-TAAATTTAAAATGTTACTTAT-3′ (SEQ ID NO: 7197) KRAS-2306 Target:5′-AAATTTAAAATGTTACTTATT-3′ (SEQ ID NO: 7198) KRAS-2307 Target:5′-AATTTAAAATGTTACTTATTT-3′ (SEQ ID NO: 7199) KRAS-2309 Target:5′-TTTAAAATGTTACTTATTTTA-3′ (SEQ ID NO: 7200) KRAS-2310 Target:5′-TTAAAATGTTACTTATTTTAA-3′ (SEQ ID NO: 7201) KRAS-2311 Target:5′-TAAAATGTTACTTATTTTAAA-3′ (SEQ ID NO: 7202) KRAS-2312 Target:5′-AAAATGTTACTTATTTTAAAA-3′ (SEQ ID NO: 7203) KRAS-2313 Target:5′-AAATGTTACTTATTTTAAAAT-3′ (SEQ ID NO: 7204) KRAS-2315 Target:5′-ATGTTACTTATTTTAAAATAA-3′ (SEQ ID NO: 7205) KRAS-2320 Target:5′-ACTTATTTTAAAATAAATGAA-3′ (SEQ ID NO: 7206) KRAS-2322 Target:5′-TTATTTTAAAATAAATGAAGT-3′ (SEQ ID NO: 7207) KRAS-2323 Target:5′-TATTTTAAAATAAATGAAGTG-3′ (SEQ ID NO: 7208) KRAS-2326 Target:5′-TTTAAAATAAATGAAGTGAGA-3′ (SEQ ID NO: 7209) KRAS-2327 Target:5′-TTAAAATAAATGAAGTGAGAT-3′ (SEQ ID NO: 7210) KRAS-2447 Target:5′-TCCATTTCTTCATGTTAAAAG-3′ (SEQ ID NO: 7211) KRAS-2448 Target:5′-CCATTTCTTCATGTTAAAAGA-3′ (SEQ ID NO: 7212) KRAS-2475 Target:5′-CTCAAACTCTTAGTTTTTTTT-3′ (SEQ ID NO: 7213) KRAS-2485 Target:5′-TAGTTTTTTTTTTTTACAACT-3′ (SEQ ID NO: 7214) KRAS-2486 Target:5′-AGTTTTTTTTTTTTACAACTA-3′ (SEQ ID NO: 7215) KRAS-2487 Target:5′-GTTTTTTTTTTTTACAACTAT-3′ (SEQ ID NO: 7216) KRAS-2488 Target:5′-TTTTTTTTTTTTACAACTATG-3′ (SEQ ID NO: 7217) KRAS-2489 Target:5′-TTTTTTTTTTTACAACTATGT-3′ (SEQ ID NO: 7218) KRAS-2490 Target:5′-TTTTTTTTTTACAACTATGTA-3′ (SEQ ID NO: 7219) KRAS-2491 Target:5′-TTTTTTTTTACAACTATGTAA-3′ (SEQ ID NO: 7220) KRAS-2492 Target:5′-TTTTTTTTACAACTATGTAAT-3′ (SEQ ID NO: 7221) KRAS-2493 Target:5′-TTTTTTTACAACTATGTAATT-3′ (SEQ ID NO: 7222) KRAS-2494 Target:5′-TTTTTTACAACTATGTAATTT-3′ (SEQ ID NO: 7223) KRAS-2495 Target:5′-TTTTTACAACTATGTAATTTA-3′ (SEQ ID NO: 7224) KRAS-2502 Target:5′-AACTATGTAATTTATATTCCA-3′ (SEQ ID NO: 7225) KRAS-2503 Target:5′-ACTATGTAATTTATATTCCAT-3′ (SEQ ID NO: 7226) KRAS-2504 Target:5′-CTATGTAATTTATATTCCATT-3′ (SEQ ID NO: 7227) KRAS-2510 Target:5′-AATTTATATTCCATTTACATA-3′ (SEQ ID NO: 7228) KRAS-2511 Target:5′-ATTTATATTCCATTTACATAA-3′ (SEQ ID NO: 7229) KRAS-2512 Target:5′-TTTATATTCCATTTACATAAG-3′ (SEQ ID NO: 7230) KRAS-2513 Target:5′-TTATATTCCATTTACATAAGG-3′ (SEQ ID′NO: 7231) KRAS-2525 Target:5′-TACATAAGGATACACTTATTT-3′ (SEQ ID NO: 7232) KRAS-2529 Target:5′-TAAGGATACACTTATTTGTCA-3′ (SEQ ID NO: 7233) KRAS-2561 Target:5′-ATCTGTAAATTTTTAACCTAT-3′ (SEQ ID NO: 7234) KRAS-2562 Target:5′-TCTGTAAATTTTTAACCTATG-3′ (SEQ ID NO: 7235) KRAS-2563 Target:5′-CTGTAAATTTTTAACCTATGT-3′ (SEQ ID NO: 7236) KRAS-2619 Target:5′-TGCAAGAGGTGAAGTTTATAT-3′ (SEQ ID NO: 7237) KRAS-2621 Target:5′-CAAGAGGTGAAGTTTATATTT-3′ (SEQ ID NO: 7238) KRAS-2622 Target:5′-AAGAGGTGAAGTTTATATTTG-3′ (SEQ ID NO: 7239) KRAS-2624 Target:5′-GAGGTGAAGTTTATATTTGAA-3′ (SEQ ID NO: 7240) KRAS-2625 Target:5′-AGGTGAAGTTTATATTTGAAT-3′ (SEQ ID NO: 7241) KRAS-2630 Target:5′-AAGTTTATATTTGAATATCCA-3′ (SEQ ID NO: 7242) KRAS-2718 Target:5′-ACTTGATGCAGTTTTAATACT-3′ (SEQ ID NO: 7243) KRAS-2720 Target:5′-TTGATGCAGTTTTAATACTTG-3′ (SEQ ID NO: 7244) KRAS-2871 Target:5′-GATTTGACCTAATCACTAATT-3′ (SEQ ID NO: 7245) KRAS-2877 Target:5′-ACCTAATCACTAATTTTCAGG-3′ (SEQ ID NO: 7246) KRAS-2946 Target:5′-CAGTAGGATTTTTCAAACCTG-3′ (SEQ ID NO: 7247) KRAS-2991 Target:5′-AGTGGAAGGAGAATTTAATAA-3′ (SEQ ID NO: 7248) KRAS-2994 Target:5′-GGAAGGAGAATTTAATAAAGA-3′ (SEQ ID NO: 7249) KRAS-2996 Target:5′-AAGGAGAATTTAATAAAGATA-3′ (SEQ ID NO: 7250) KRAS-2997 Target:5′-AGGAGAATTTAATAAAGATAG-3′ (SEQ ID NO: 7251) KRAS-3001 Target:5′-GAATTTAATAAAGATAGTGCT-3′ (SEQ ID NO: 7252) KRAS-3030 Target:5′-TCCTTAGGTAATCTATAACTA-3′ (SEQ ID NO: 7253) KRAS-3031 Target:5′-CCTTAGGTAATCTATAACTAG-3′ (SEQ ID NO: 7254) KRAS-3065 Target:5′-AACAGTAATACATTCCATTGT-3′ (SEQ ID NO: 7255) KRAS-3067 Target:5′-CAGTAATACATTCCATTGTTT-3′ (SEQ ID NO: 7256) KRAS-3068 Target:5′-AGTAATACATTCCATTGTTTT-3′ (SEQ ID NO: 7257) KRAS-3069 Target:5′-GTAATACATTCCATTGTTTTA-3′ (SEQ ID NO: 7258) KRAS-3079 Target:5′-CCATTGTTTTAGTAACCAGAA-3′ (SEQ ID NO: 7259) KRAS-3093 Target:5′-ACCAGAAATCTTCATGCAATG-3′ (SEQ ID NO: 7260) KRAS-3103 Target:5′-TTCATGCAATGAAAAATACTT-3′ (SEQ ID NO: 7261) KRAS-3110 Target:5′-AATGAAAAATACTTTAATTCA-3′ (SEQ ID NO: 7262) KRAS-3112 Target:5′-TGAAAAATACTTTAATTCATG-3′ (SEQ ID NO: 7263) KRAS-3113 Target:5′-GAAAAATACTTTAATTCATGA-3′ (SEQ ID NO: 7264) KRAS-3127 Target:5′-TTCATGAAGCTTACTTTTTTT-3′ (SEQ ID NO: 7265) KRAS-3130 Target:5′-ATGAAGCTTACTTTTTTTTTT-3′ (SEQ ID NO: 7266) KRAS-3134 Target:5′-AGCTTACTTTTTTTTTTTGGT-3′ (SEQ ID NO: 7267) KRAS-3138 Target:5′-TACTTTTTTTTTTTGGTGTCA-3′ (SEQ ID NO: 7268) KRAS-3139 Target:5′-ACTTTTTTTTTTTGGTGTCAG-3′ (SEQ ID NO: 7269) KRAS-3297 Target:5′-AACTAATTTTTGTATTTTTAG-3′ (SEQ ID NO: 7270) KRAS-3300 Target:5′-TAATTTTTGTATTTTTAGGAG-3′ (SEQ ID NO: 7271) KRAS-3413 Target:5′-CTCATTTATTCAGCAAATATT-3′ (SEQ ID NO: 7272) KRAS-3415 Target:5′-CATTTATTCAGCAAATATTTA-3′ (SEQ ID NO: 7273) KRAS-3417 Target:5′-TTTATTCAGCAAATATTTATT-3′ (SEQ ID NO: 7274) KRAS-3589 Target:5′-TATTTTAGTTTTGCAAAGAAG-3′ (SEQ ID NO: 7275) KRAS-3630 Target:5′-CTCTATAATTGTTTTGCTACG-3′ (SEQ ID NO: 7276) KRAS-3677 Target:5′-TACTTTATGTAAATCACTTCA-3′ (SEQ ID NO: 7277) KRAS-3678 Target:5′-ACTTTATGTAAATCACTTCAT-3′ (SEQ ID NO: 7278) KRAS-3679 Target:5′-CTTTATGTAAATCACTTCATT-3′ (SEQ ID NO: 7279) KRAS-3680 Target:5′-TTTATGTAAATCACTTCATTG-3′ (SEQ ID NO: 7280) KRAS-3681 Target:5′-TTATGTAAATCACTTCATTGT-3′ (SEQ ID NO: 7281) KRAS-3682 Target:5′-TATGTAAATCACTTCATTGTT-3′ (SEQ ID NO: 7282) KRAS-3683 Target:5′-ATGTAAATCACTTCATTGTTT-3′ (SEQ ID NO: 7283) KRAS-3697 Target:5′-ATTGTTTTAAAGGAATAAACT-3′ (SEQ ID NO: 7284) KRAS-3698 Target:5′-TTGTTTTAAAGGAATAAACTT-3′ (SEQ ID NO: 7285) KRAS-3701 Target:5′-TTTTAAAGGAATAAACTTGAT-3′ (SEQ ID NO: 7286) KRAS-3702 Target:5′-TTTAAAGGAATAAACTTGATT-3′ (SEQ ID NO: 7287) KRAS-3703 Target:5′-TTAAAGGAATAAACTTGATTA-3′ (SEQ ID NO: 7288) KRAS-3705 Target:5′-AAAGGAATAAACTTGATTATA-3′ (SEQ ID NO: 7289) KRAS-3706 Target:5′-AAGGAATAAACTTGATTATAT-3′ (SEQ ID NO: 7290) KRAS-3707 Target:5′-AGGAATAAACTTGATTATATT-3′ (SEQ ID NO: 7291) KRAS-3708 Target:5′-GGAATAAACTTGATTATATTG-3′ (SEQ ID NO: 7292) KRAS-3709 Target:5′-GAATAAACTTGATTATATTGT-3′ (SEQ ID NO: 7293) KRAS-3714 Target:5′-AACTTGATTATATTGTTTTTT-3′ (SEQ ID NO: 7294) KRAS-3715 Target:5′-ACTTGATTATATTGTTTTTTT-3′ (SEQ ID NO: 7295) KRAS-3718 Target:5′-TGATTATATTGTTTTTTTATT-3′ (SEQ ID NO: 7296) KRAS-3723 Target:5′-ATATTGTTTTTTTATTTGGCA-3′ (SEQ ID NO: 7297) KRAS-3724 Target:5′-TATTGTTTTTTTATTTGGCAT-3′ (SEQ ID NO: 7298) KRAS-3728 Target:5′-GTTTTTTTATTTGGCATAACT-3′ (SEQ ID NO: 7299) KRAS-3729 Target:5′-TTTTTTTATTTGGCATAACTG-3′ (SEQ ID NO: 7300) KRAS-3741 Target:5′-GCATAACTGTGATTCTTTTAG-3′ (SEQ ID NO: 7301) KRAS-3746 Target:5′-ACTGTGATTCTTTTAGGACAA-3′ (SEQ ID NO: 7302) KRAS-3747 Target:5′-CTGTGATTCTTTTAGGACAAT-3′ (SEQ ID NO: 7303) KRAS-3783 Target:5′-GGTGTATGTCAGATATTCATA-3′ (SEQ ID NO: 7304) KRAS-3784 Target:5′-GTGTATGTCAGATATTCATAT-3′ (SEQ ID NO: 7305) KRAS-3810 Target:5′-CAAATGTGTAATATTCCAGTT-3′ (SEQ ID NO: 7306) KRAS-3838 Target:5′-CATAAGTAATTAAAATATACT-3′ (SEQ ID NO: 7307) KRAS-3839 Target:5′-ATAAGTAATTAAAATATACTT-3′ (SEQ ID NO: 7308) KRAS-3840 Target:5′-TAAGTAATTAAAATATACTTA-3′ (SEQ ID NO: 7309) KRAS-3841 Target:5′-AAGTAATTAAAATATACTTAA-3′ (SEQ ID NO: 7310) KRAS-3842 Target:5′-AGTAATTAAAATATACTTAAA-3′ (SEQ ID NO: 7311) KRAS-3843 Target:5′-GTAATTAAAATATACTTAAAA-3′ (SEQ ID NO: 7312) KRAS-3844 Target:5′-TAATTAAAATATACTTAAAAA-3′ (SEQ ID NO: 7313) KRAS-3845 Target:5′-AATTAAAATATACTTAAAAAT-3′ (SEQ ID NO: 7314) KRAS-3846 Target:5′-ATTAAAATATACTTAAAAATT-3′ (SEQ ID NO: 7315) KRAS-3848 Target:5′-TAAAATATACTTAAAAATTAA-3′ (SEQ ID NO: 7316) KRAS-3849 Target:5′-AAAATATACTTAAAAATTAAT-3′ (SEQ ID NO: 7317) KRAS-3850 Target:5′-AAATATACTTAAAAATTAATA-3′ (SEQ ID NO: 7318) KRAS-3851 Target:5′-AATATACTTAAAAATTAATAG-3′ (SEQ ID NO: 7319) KRAS-3855 Target:5′-TACTTAAAAATTAATAGTTTT-3′ (SEQ ID NO: 7320) KRAS-3859 Target:5′-TAAAAATTAATAGTTTTATCT-3′ (SEQ ID NO: 7321) KRAS-3860 Target:5′-AAAAATTAATAGTTTTATCTG-3′ (SEQ ID NO: 7322) KRAS-3861 Target:5′-AAAATTAATAGTTTTATCTGG-3′ (SEQ ID NO: 7323) KRAS-3876 Target:5′-ATCTGGGTACAAATAAACAGG-3′ (SEQ ID NO: 7324) KRAS-3915 Target:5′-GACAAGGAAACTTCTATGTAA-3′ (SEQ ID NO: 7325) KRAS-3916 Target:5′-ACAAGGAAACTTCTATGTAAA-3′ (SEQ ID NO: 7326) KRAS-3917 Target:5′-CAAGGAAACTTCTATGTAAAA-3′ (SEQ ID NO: 7327) KRAS-3926 Target:5′-TTCTATGTAAAAATCACTATG-3′ (SEQ ID NO: 7328) KRAS-3927 Target:5′-TCTATGTAAAAATCACTATGA-3′ (SEQ ID NO: 7329) KRAS-3928 Target:5′-CTATGTAAAAATCACTATGAT-3′ (SEQ ID NO: 7330) KRAS-3932 Target:5′-GTAAAAATCACTATGATTTCT-3′ (SEQ ID NO: 7331) KRAS-3933 Target:5′-TAAAAATCACTATGATTTCTG-3′ (SEQ ID NO: 7332) KRAS-3942 Target:51-CTATGATTTCTGAATTGCTAT-3′ (SEQ ID NO: 7333) KRAS-3943 Target:5′-TATGATTTCTGAATTGCTATG-3′ (SEQ ID NO: 7334) KRAS-3960 Target:5′-TATGTGAAACTACAGATCTTT-3′ (SEQ ID NO: 7335) KRAS-3961 Target:5′-ATGTGAAACTACAGATCTTTG-3′ (SEQ ID NO: 7336) KRAS-3997 Target:5′-AGGGTGTTAAGACTTACACAG-3′ (SEQ ID NO: 7337) KRAS-4084 Target:5′-TTTAGGCCTCTTGAATTTTTG-3′ (SEQ ID NO: 7338) KRAS-4092 Target:5′-TCTTGAATTTTTGATGTAGAT-3′ (SEQ ID NO: 7339) KRAS-4093 Target:5′-CTTGAATTTTTGATGTAGATG-3′ (SEQ ID NO: 7340) KRAS-4108 Target:5′-TAGATGGGCATTTTTTTAAGG-3′ (SEQ ID NO: 7341) KRAS-4114 Target:5′-GGCATTTTTTTAAGGTAGTGG-3′ (SEQ ID NO: 7342) KRAS-4126 Target:5′-AGGTAGTGGTTAATTACCTTT-3′ (SEQ ID NO: 7343) KRAS-4128 Target:5′-GTAGTGGTTAATTACCTTTAT-3′ (SEQ ID NO: 7344) KRAS-4129 Target:5′-TAGTGGTTAATTACCTTTATG-3′ (SEQ ID NO: 7345) KRAS-4130 Target:5′-AGTGGTTAATTACCTTTATGT-3′ (SEQ ID NO: 7346) KRAS-4131 Target:5′-GTGGTTAATTACCTTTATGTG-3′ (SEQ ID NO: 7347) KRAS-4141 Target:5′-ACCTTTATGTGAACTTTGAAT-3′ (SEQ ID NO: 7348) KRAS-4146 Target:5′-TATGTGAACTTTGAATGGTTT-3′ (SEQ ID NO: 7349) KRAS-4152 Target:5′-AACTTTGAATGGTTTAACAAA-3′ (SEQ ID NO: 7350) KRAS-4156 Target:5′-TTGAATGGTTTAACAAAAGAT-3′ (SEQ ID NO: 7351) KRAS-4157 Target:5′-TGAATGGTTTAACAAAAGATT-3′ (SEQ ID NO: 7352) KRAS-4158 Target:5′-GAATGGTTTAACAAAAGATTT-3′ (SEQ ID NO: 7353) KRAS-4159 Target:5′-AATGGTTTAACAAAAGATTTG-3′ (SEQ ID NO: 7354) KRAS-4160 Target:5′-ATGGTTTAACAAAAGATTTGT-3′ (SEQ ID NO: 7355) KRAS-4162 Target:5′-GGTTTAACAAAAGATTTGTTT-3′ (SEQ ID NO: 7356) KRAS-4163 Target:5′-GTTTAACAAAAGATTTGTTTT-3′ (SEQ ID NO: 7357) KRAS-4167 Target:5′-AACAAAAGATTTGTTTTTGTA-3′ (SEQ ID NO: 7358) KRAS-4168 Target:5′-ACAAAAGATTTGTTTTTGTAG-3′ (SEQ ID NO: 7359) KRAS-4169 Target:5′-CAAAAGATTTGTTTTTGTAGA-3′ (SEQ ID NO: 7360) KRAS-4171 Target:5′-AAAGATTTGTTTTTGTAGAGA-3′ (SEQ ID NO: 7361) KRAS-4172 Target:5′-AAGATTTGTTTTTGTAGAGAT-3′ (SEQ ID NO: 7362) KRAS-4173 Target:5′-AGATTTGTTTTTGTAGAGATT-3′ (SEQ ID NO: 7363) KRAS-4174 Target:5′-GATTTGTTTTTGTAGAGATTT-3′ (SEQ ID NO: 7364) KRAS-4180 Target:5′-TTTTTGTAGAGATTTTAAAGG-3′ (SEQ ID NO: 7365) KRAS-4197 Target:5′-AAGGGGGAGAATTCTAGAAAT-3′ (SEQ ID NO: 7366) KRAS-4199 Target:5′-GGGGGAGAATTCTAGAAATAA-3′ (SEQ ID NO: 7367) KRAS-4200 Target:5′-GGGGAGAATTCTAGAAATAAA-3′ (SEQ ID NO: 7368) KRAS-4201 Target:5′-GGGAGAATTCTAGAAATAAAT-3′ (SEQ ID NO: 7369) KRAS-4202 Target:5′-GGAGAATTCTAGAAATAAATG-3′ (SEQ ID NO: 7370) KRAS-4203 Target:5′-GAGAATTCTAGAAATAAATGT-3′ (SEQ ID NO: 7371) KRAS-4208 Target:5′-TTCTAGAAATAAATGTTACCT-3′ (SEQ ID NO: 7372) KRAS-4209 Target:5′-TCTAGAAATAAATGTTACCTA-3′ (SEQ ID NO: 7373) KRAS-4210 Target:5′-CTAGAAATAAATGTTACCTAA-3′ (SEQ ID NO: 7374) KRAS-4211 Target:5′-TAGAAATAAATGTTACCTAAT-3′ (SEQ ID NO: 7375) KRAS-4212 Target:5′-AGAAATAAATGTTACCTAATT-3′ (SEQ ID NO: 7376) KRAS-4214 Target:5′-AAATAAATGTTACCTAATTAT-3′ (SEQ ID NO: 7377) KRAS-4225 Target:5′-ACCTAATTATTACAGCCTTAA-3′ (SEQ ID NO: 7378) KRAS-4226 Target:5′-CCTAATTATTACAGCCTTAAA-3′ (SEQ ID NO: 7379) KRAS-4240 Target:5′-CCTTAAAGACAAAAATCCTTG-3′ (SEQ ID NO: 7380) KRAS-4243 Target:5′-TAAAGACAAAAATCCTTGTTG-3′ (SEQ ID NO: 7381) KRAS-4255 Target:5′-TCCTTGTTGAAGTTTTTTTAA-3′ (SEQ ID NO: 7382) KRAS-4256 Target:5′-CCTTGTTGAAGTTTTTTTAAA-3′ (SEQ ID NO: 7383) KRAS-4258 Target:5′-TTGTTGAAGTTTTTTTAAAAA-3′ (SEQ ID NO: 7384) KRAS-4259 Target:5′-TGTTGAAGTTTTTTTAAAAAA-3′ (SEQ ID NO: 7385) KRAS-4263 Target:5′-GAAGTTTTTTTAAAAAAAGCT-3′ (SEQ ID NO: 7386) KRAS-4264 Target:5′-AAGTTTTTTTAAAAAAAGCTA-3′ (SEQ ID NO: 7387) KRAS-4265 Target:5′-AGTTTTTTTAAAAAAAGCTAA-3′ (SEQ ID NO: 7388) KRAS-4266 Target:5′-GTTTTTTTAAAAAAAGCTAAA-3′ (SEQ ID NO: 7389) KRAS-4267 Target:5′-TTTTTTTAAAAAAAGCTAAAT-3′ (SEQ ID NO: 7390) KRAS-4271 Target:5′-TTTAAAAAAAGCTAAATTACA-3′ (SEQ ID NO: 7391) KRAS-4273 Target:5′-TAAAAAAAGCTAAATTACATA-3′ (SEQ ID NO: 7392) KRAS-4295 Target:5′-ACTTAGGCATTAACATGTTTG-3′ (SEQ ID NO: 7393) KRAS-4296 Target:5′-CTTAGGCATTAACATGTTTGT-3′ (SEQ ID NO: 7394) KRAS-4327 Target:5′-AGCAGACGTATATTGTATCAT-3′ (SEQ ID NO: 7395) KRAS-4331 Target:5′-GACGTATATTGTATCATTTGA-3′ (SEQ ID NO: 7396) KRAS-4333 Target:5′-CGTATATTGTATCATTTGAGT-3′ (SEQ ID NO: 7397) KRAS-4336 Target:5′-ATATTGTATCATTTGAGTGAA-3′ (SEQ ID NO: 7398) KRAS-4337 Target:5′-TATTGTATCATTTGAGTGAAT-3′ (SEQ ID NO: 7399) KRAS-4338 Target:5′-ATTGTATCATTTGAGTGAATG-3′ (SEQ ID NO: 7400) KRAS-4404 Target:5′-ATAGGAATTTAGAACCTAACT-3′ (SEQ ID NO: 7401) KRAS-4405 Target:5′-TAGGAATTTAGAACCTAACTT-3′ (SEQ ID NO: 7402) KRAS-4409 Target:5′-AATTTAGAACCTAACTTTTAT-3′ (SEQ ID NO: 7403) KRAS-4410 Target:5′-ATTTAGAACCTAACTTTTATA-3′ (SEQ ID NO: 7404) KRAS-4411 Target:5′-TTTAGAACCTAACTTTTATAG-3′ (SEQ ID NO: 7405) KRAS-4412 Target:5′-TTAGAACCTAACTTTTATAGG-3′ (SEQ ID NO: 7406) KRAS-4424 Target:5′-TTTTATAGGTTATCAAAACTG-3′ (SEQ ID NO: 7407) KRAS-4426 Target:5′-TTATAGGTTATCAAAACTGTT-3′ (SEQ ID NO: 7408) KRAS-4460 Target:5′-AATTTTGTCCTAATATATACA-3′ (SEQ ID NO: 7409) KRAS-4461 Target:5′-ATTTTGTCCTAATATATACAT-3′ (SEQ ID NO: 7410) KRAS-4462 Target:5′-TTTTGTCCTAATATATACATA-3′ (SEQ ID NO: 7411) KRAS-4468 Target:5′-CCTAATATATACATAGAAACT-3′ (SEQ ID NO: 7412) KRAS-4473 Target:5′-TATATACATAGAAACTTTGTG-3′ (SEQ ID NO: 7413) KRAS-4516 Target:5′-CACAAGTTCATCTCATTTGTA-3′ (SEQ ID NO: 7414) KRAS-4527 Target:5′-CTCATTTGTATTCCATTGATT-3′ (SEQ ID NO: 7415) KRAS-4528 Target:5′-TCATTTGTATTCCATTGATTT-3′ (SEQ ID NO: 7416) KRAS-4529 Target:5′-CATTTGTATTCCATTGATTTT-3′ (SEQ ID NO: 7417) KRAS-4530 Target:5′-ATTTGTATTCCATTGATTTTT-3′ (SEQ ID NO: 7418) KRAS-4531 Target:5′-TTTGTATTCCATTGATTTTTT-3′ (SEQ ID NO: 7419) KRAS-4532 Target:5′-TTGTATTCCATTGATTTTTTT-3′ (SEQ ID NO: 7420) KRAS-4533 Target:5′-TGTATTCCATTGATTTTTTTT-3′ (SEQ ID NO: 7421) KRAS-4540 Target:5′-CATTGATTTTTTTTTTCTTCT-3′ (SEQ ID NO: 7422) KRAS-4541 Target:5′-ATTGATTTTTTTTTTCTTCTA-3′ (SEQ ID NO: 7423) KRAS-4545 Target:5′-ATTTTTTTTTTCTTCTAAACA-3′ (SEQ ID NO: 7424) KRAS-4546 Target:5′-TTTTTTTTTTCTTCTAAACAT-3′ (SEQ ID NO: 7425) KRAS-4547 Target:5′-TTTTTTTTTCTTCTAAACATT-3′ (SEQ ID NO: 7426) KRAS-4548 Target:5′-TTTTTTTTCTTCTAAACATTT-3′ (SEQ ID NO: 7427) KRAS-4549 Target:5′-TTTTTTTCTTCTAAACATTTT-3′ (SEQ ID NO: 7428) KRAS-4562 Target:5′-AACATTTTTTCTTCAAACAGT-3′ (SEQ ID NO: 7429) KRAS-4563 Target:5′-ACATTTTTTCTTCAAACAGTA-3′ (SEQ ID NO: 7430) KRAS-4564 Target:5′-CATTTTTTCTTCAAACAGTAT-3′ (SEQ ID NO: 7431) KRAS-4565 Target:5′-ATTTTTTCTTCAAACAGTATA-3′ (SEQ ID NO: 7432) KRAS-4566 Target:5′-TTTTTTCTTCAAACAGTATAT-3′ (SEQ ID NO: 7433) KRAS-4573 Target:5′-TTCAAACAGTATATAACTTTT-3′ (SEQ ID NO: 7434) KRAS-4578 Target:5′-ACAGTATATAACTTTTTTTAG-3′ (SEQ ID NO: 7435) KRAS-4579 Target:5′-CAGTATATAACTTTTTTTAGG-3′ (SEQ ID NO: 7436) KRAS-4580 Target:5′-AGTATATAACTTTTTTTAGGG-3′ (SEQ ID NO: 7437) KRAS-4581 Target:5′-GTATATAACTTTTTTTAGGGG-3′ (SEQ ID NO: 7438) KRAS-4587 Target:5′-AACTTTTTTTAGGGGATTTTT-3′ (SEQ ID NO: 7439) KRAS-4588 Target:5′-ACTTTTTTTAGGGGATTTTTT-3′ (SEQ ID NO: 7440) KRAS-4599 Target:5′-GGGATTTTTTTTTAGACAGCA-3′ (SEQ ID NO: 7441) KRAS-4600 Target:5′-GGATTTTTTTTTAGACAGCAA-3′ (SEQ ID NO: 7442) KRAS-4601 Target:5′-GATTTTTTTTTAGACAGCAAA-3′ (SEQ ID NO: 7443) KRAS-4629 Target:5′-TGAAGATTTCCATTTGTCAAA-3′ (SEQ ID NO: 7444) KRAS-4630 Target:5′-GAAGATTTCCATTTGTCAAAA-3′ (SEQ ID NO: 7445) KRAS-4631 Target:5′-AAGATTTCCATTTGTCAAAAA-3′ (SEQ ID NO: 7446) KRAS-4632 Target:5′-AGATTTCCATTTGTCAAAAAG-3′ (SEQ ID NO: 7447) KRAS-4637 Target:5′-TCCATTTGTCAAAAAGTAATG-3′ (SEQ ID NO: 7448) KRAS-4638 Target:5′-CCATTTGTCAAAAAGTAATGA-3′ (SEQ ID NO: 7449) KRAS-4639 Target:5′-CATTTGTCAAAAAGTAATGAT-3′ (SEQ ID NO: 7450) KRAS-4644 Target:5′-GTCAAAAAGTAATGATTTCTT-3′ (SEQ ID NO: 7451) KRAS-4645 Target:5′-TCAAAAAGTAATGATTTCTTG-3′ (SEQ ID NO: 7452) KRAS-4646 Target:5′-CAAAAAGTAATGATTTCTTGA-3′ (SEQ ID NO: 7453) KRAS-4647 Target:5′-AAAAAGTAATGATTTCTTGAT-3′ (SEQ ID NO: 7454) KRAS-4649 Target:5′-AAAGTAATGATTTCTTGATAA-3′ (SEQ ID NO: 7455) KRAS-4651 Target:5′-AGTAATGATTTCTTGATAATT-3′ (SEQ ID NO: 7456) KRAS-4652 Target:5′-GTAATGATTTCTTGATAATTG-3′ (SEQ ID NO: 7457) KRAS-4654 Target:5′-AATGATTTCTTGATAATTGTG-3′ (SEQ ID NO: 7458) KRAS-4655 Target:5′-ATGATTTCTTGATAATTGTGT-3′ (SEQ ID NO: 7459) KRAS-4660 Target:5′-TTCTTGATAATTGTGTAGTAA-3′ (SEQ ID NO: 7460) KRAS-4661 Target:5′-TCTTGATAATTGTGTAGTAAT-3′ (SEQ ID NO: 7461) KRAS-4662 Target:5′-CTTGATAATTGTGTAGTAATG-3′ (SEQ ID NO: 7462) KRAS-4664 Target:5′-TGATAATTGTGTAGTAATGTT-3′ (SEQ ID NO: 7463) KRAS-4667 Target:5′-TAATTGTGTAGTAATGTTTTT-3′ (SEQ ID NO: 7464) KRAS-4669 Target:5′-ATTGTGTAGTAATGTTTTTTA-3′ (SEQ ID NO: 7465) KRAS-4670 Target:5′-TTGTGTAGTAATGTTTTTTAG-3′ (SEQ ID NO: 7466) KRAS-4704 Target:5′-CCTTAAAGCTGAATTTATATT-3′ (SEQ ID NO: 7467) KRAS-4706 Target:5′-TTAAAGCTGAATTTATATTTA-3′ (SEQ ID NO: 7468) KRAS-4715 Target:5′-AATTTATATTTAGTAACTTCT-3′ (SEQ ID NO: 7469) KRAS-4716 Target:5′-ATTTATATTTAGTAACTTCTG-3′ (SEQ ID NO: 7470) KRAS-4717 Target:5′-TTTATATTTAGTAACTTCTGT-3′ (SEQ ID NO: 7471) KRAS-4722 Target:5′-ATTTAGTAACTTCTGTGTTAA-3′ (SEQ ID NO: 7472) KRAS-4773 Target:5′-ACTGAATAGCTGTCATAAAAT-3′ (SEQ ID NO: 7473) KRAS-4774 Target:5′-CTGAATAGCTGTCATAAAATG-3′ (SEQ ID NO: 7474) KRAS-4788 Target:5′-TAAAATGAAACTTTCTTTCTA-3′ (SEQ ID NO: 7475) KRAS-4789 Target:5′-AAAATGAAACTTTCTTTCTAA-3′ (SEQ ID NO: 7476) KRAS-4790 Target:5′-AAATGAAACTTTCTTTCTAAA-3′ (SEQ ID NO: 7477) KRAS-4792 Target:5′-ATGAAACTTTCTTTCTAAAGA-3′ (SEQ ID NO: 7478) KRAS-4796 Target:5′-AACTTTCTTTCTAAAGAAAGA-3′ (SEQ ID NO: 7479) KRAS-4821 Target:5′-CACATGAGTTCTTGAAGAATA-3′ (SEQ ID NO: 7480) KRAS-4830 Target:5′-TCTTGAAGAATAGTCATAACT-3′ (SEQ ID NO:.7481) KRAS-4831 Target:5′-CTTGAAGAATAGTCATAACTA-3′ (SEQ ID NO: 7482) KRAS-4832 Target:5′-TTGAAGAATAGTCATAACTAG-3′ (SEQ ID NO: 7483) KRAS-4835 Target:5′-AAGAATAGTCATAACTAGATT-3′ (SEQ ID NO: 7484) KRAS-4836 Target:5′-AGAATAGTCATAACTAGATTA-3′ (SEQ ID NO: 7485) KRAS-4837 Target:5′-GAATAGTCATAACTAGATTAA-3′ (SEQ ID NO: 7486) KRAS-4838 Target:5′-AATAGTCATAACTAGATTAAG-3′ (SEQ ID NO: 7487) KRAS-4842 Target:5′-GTCATAACTAGATTAAGATCT-3′ (SEQ ID NO: 7488) KRAS-4848 Target:5′-ACTAGATTAAGATCTGTGTTT-3′ (SEQ ID NO: 7489) KRAS-4850 Target:5′-TAGATTAAGATCTGTGTTTTA-3′ (SEQ ID NO: 7490) KRAS-4851 Target:5′-AGATTAAGATCTGTGTTTTAG-3′ (SEQ ID NO: 7491) KRAS-4853 Target:5′-ATTAAGATCTGTGTTTTAGTT-3′ (SEQ ID NO: 7492) KRAS-4860 Target:5′-TCTGTGTTTTAGTTTAATAGT-3′ (SEQ ID NO: 7493) KRAS-4861 Target:5′-CTGTGTTTTAGTTTAATAGTT-3′ (SEQ ID NO: 7494) KRAS-4863 Target:5′-GTGTTTTAGTTTAATAGTTTG-3′ (SEQ ID NO: 7495) KRAS-4865 Target:5′-GTTTTAGTTTAATAGTTTGAA-3′ (SEQ ID NO: 7496) KRAS-4866 Target:5′-TTTTAGTTTAATAGTTTGAAG-3′ (SEQ ID NO: 7497) KRAS-4892 Target:5′-GTTTGGGATAATGATAGGTAA-3′ (SEQ ID NO: 7498) KRAS-4894 Target:5′-TTGGGATAATGATAGGTAATT-3′ (SEQ ID NO: 7499) KRAS-4897 Target:5′-GGATAATGATAGGTAATTTAG-3′ (SEQ ID NO: 7500) KRAS-4898 Target:5′-GATAATGATAGGTAATTTAGA-3′ (SEQ ID NO: 7501) KRAS-4901 Target:5′-AATGATAGGTAATTTAGATGA-3′ (SEQ ID NO: 7502) KRAS-4902 Target:5′-ATGATAGGTAATTTAGATGAA-3′ (SEQ ID NO: 7503) KRAS-4907 Target:5′-AGGTAATTTAGATGAATTTAG-3′ (SEQ ID NO: 7504) KRAS-4909 Target:5′-GTAATTTAGATGAATTTAGGG-3′ (SEQ ID NO: 7505) KRAS-4916 Target:5′-AGATGAATTTAGGGGAAAAAA-3′ (SEQ ID NO: 7506) KRAS-4917 Target:5′-GATGAATTTAGGGGAAAAAAA-3′ (SEQ ID NO: 7507) KRAS-4928 Target:5′-GGGAAAAAAAAGTTATCTGCA-3′ (SEQ ID NO: 7508) KRAS-4929 Target:5′-GGAAAAAAAAGTTATCTGCAG-3′ (SEQ ID NO: 7509) KRAS-4934 Target:5′-AAAAAGTTATCTGCAGATATG-3′ (SEQ ID NO: 7510) KRAS-5035 Target:5′-GTCTTGTGTTTTCATGTTGAA-3′ (SEQ ID NO: 7511) KRAS-5036 Target:5′-TCTTGTGTTTTCATGTTGAAA-3′ (SEQ ID NO: 7512) KRAS-5037 Target:5′-CTTGTGTTTTCATGTTGAAAA-3′ (SEQ ID NO: 7513) KRAS-5047 Target:5′-CATGTTGAAAATACTTTTGCA-3′ (SEQ ID NO: 7514) KRAS-5048 Target:5′-ATGTTGAAAATACTTTTGCAT-3′ (SEQ ID NO: 7515) KRAS-5049 Target:5′-TGTTGAAAATACTTTTGCATT-3′ (SEQ ID NO: 7516) KRAS-5050 Target:5′-GTTGAAAATACTTTTGCATTT-3′ (SEQ ID NO: 7517) KRAS-5060 Target:5′-CTTTTGCATTTTTCCTTTGAG-3′ (SEQ ID NO: 7518) KRAS-5077 Target:5′-TGAGTGCCAATTTCTTACTAG-3′ (SEQ ID NO: 7519) KRAS-5082 Target:5′-GCCAATTTCTTACTAGTACTA-3′ (SEQ ID NO: 7520) KRAS-5083 Target:5′-CCAATTTCTTACTAGTACTAT-3′ (SEQ ID NO: 7521) KRAS-5092 Target:5′-TACTAGTACTATTTCTTAATG-3′ (SEQ ID NO: 7522) KRAS-5093 Target:5′-ACTAGTACTATTTCTTAATGT-3′ (SEQ ID NO: 7523) KRAS-5098 Target:5′-TACTATTTCTTAATGTAACAT-3′ (SEQ ID NO: 7524) KRAS-5099 Target:5′-ACTATTTCTTAATGTAACATG-3′ (SEQ ID NO: 7525) KRAS-5100 Target:5′-CTATTTCTTAATGTAACATGT-3′ (SEQ ID NO: 7526) KRAS-5107 Target:5′-TTAATGTAACATGTTTACCTG-3′ (SEQ ID NO: 7527) KRAS-5124 Target:5′-CCTGGAATGTATTTTAACTAT-3′ (SEQ ID NO: 7528) KRAS-5126 Target:5′-TGGAATGTATTTTAACTATTT-3′ (SEQ ID NO: 7529) KRAS-5127 Target:5′-GGAATGTATTTTAACTATTTT-3′ (SEQ ID NO: 7530) KRAS-5128 Target:5′-GAATGTATTTTAACTATTTTT-3′ (SEQ ID NO: 7531) KRAS-5129 Target:5′-AATGTATTTTAACTATTTTTG-3′ (SEQ ID NO: 7532) KRAS-5131 Target:5′-TGTATTTTAACTATTTTTGTA-3′ (SEQ ID NO: 7533) KRAS-5133 Target:5′-TATTTTAACTATTTTTGTATA-3′ (SEQ ID NO: 7534) KRAS-5134 Target:5′-ATTTTAACTATTTTTGTATAG-3′ (SEQ ID NO: 7535) KRAS-5135 Target:5′-TTTTAACTATTTTTGTATAGT-3′ (SEQ ID NO: 7536) KRAS-5142 Target:5′-TATTTTTGTATAGTGTAAACT-3′ (SEQ ID NO: 7537) KRAS-5143 Target:5′-ATTTTTGTATAGTGTAAACTG-3′ (SEQ ID NO: 7538) KRAS-5144 Target:5′-TTTTTGTATAGTGTAAACTGA-3′ (SEQ ID NO: 7539) KRAS-5159 Target:5′-AACTGAAACATGCACATTTTG-3′ (SEQ ID NO: 7540) KRAS-5165 Target:5′-AACATGCACATTTTGTACATT-3′ (SEQ ID NO: 7541) KRAS-5218 Target:5′-TCCAGTTGTTTTCCATCATTT-3′ (SEQ ID NO: 7542) KRAS-5220 Target:5′-CAGTTGTTTTCCATCATTTGG-3′ (SEQ ID NO: 7543) KRAS-5257 Target:5′-ATGTTGGTCATATCAAACATT-3′ (SEQ ID NO: 7544) KRAS-5258 Target:5′-TGTTGGTCATATCAAACATTA-3′ (SEQ ID NO: 7545) KRAS-5259 Target:5′-GTTGGTCATATCAAACATTAA-3′ (SEQ ID NO: 7546) KRAS-5263 Target:5′-GTCATATCAAACATTAAAAAT-3′ (SEQ ID NO: 7547) KRAS-5279 Target:5′-AAAATGACCACTCTTTTAATT-3′ (SEQ ID NO: 7548) KRAS-5280 Target:5′-AAATGACCACTCTTTTAATTG-3′ (SEQ ID NO: 7549) KRAS-5289 Target:5′-CTCTTTTAATTGAAATTAACT-3′ (SEQ ID NO: 7550) KRAS-5290 Target:5′-TCTTTTAATTGAAATTAACTT-3′ (SEQ ID NO: 7551) KRAS-5292 Target:5′-TTTTAATTGAAATTAACTTTT-3′ (SEQ ID NO: 7552) KRAS-5294 Target:5′-TTAATTGAAATTAACTTTTAA-3′ (SEQ ID NO: 7553) KRAS-5295 Target:5′-TAATTGAAATTAACTTTTAAA-3′ (SEQ ID NO: 7554) KRAS-5296 Target:5′-AATTGAAATTAACTTTTAAAT-3′ (SEQ ID NO: 7555) KRAS-5297 Target:5′-ATTGAAATTAACTTTTAAATG-3′ (SEQ ID NO: 7556) KRAS-5298 Target:5′-TTGAAATTAACTTTTAAATGT-3′ (SEQ ID NO: 7557) KRAS-5299 Target:5′-TGAAATTAACTTTTAAATGTT-3′ (SEQ ID NO: 7558) KRAS-5302 Target:5′-AATTAACTTTTAAATGTTTAT-3′ (SEQ ID NO: 7559) KRAS-5306 Target:5′-AACTTTTAAATGTTTATAGGA-3′ (SEQ ID NO: 7560) KRAS-5310 Target:5′-TTTAAATGTTTATAGGAGTAT-3′ (SEQ ID NO: 7561) KRAS-5311 Target:5′-TTAAATGTTTATAGGAGTATG-3′ (SEQ ID NO: 7562) KRAS-5333 Target:5′-GCTGTGAAGTGATCTAAAATT-3′ (SEQ ID NO: 7563) KRAS-5335 Target:5′-TGTGAAGTGATCTAAAATTTG-3′ (SEQ ID NO: 7564) KRAS-5336 Target:5′-GTGAAGTGATCTAAAATTTGT-3′ (SEQ ID NO: 7565) KRAS-5337 Target:5′-TGAAGTGATCTAAAATTTGTA-3′ (SEQ ID NO: 7566) KRAS-5338 Target:5′-GAAGTGATCTAAAATTTGTAA-3′ (SEQ ID NO: 7567) KRAS-5339 Target:5′-AAGTGATCTAAAATTTGTAAT-3′ (SEQ ID NO: 7568) KRAS-5340 Target:5′-AGTGATCTAAAATTTGTAATA-3′ (SEQ ID NO: 7569) KRAS-5344 Target:5′-ATCTAAAATTTGTAATATTTT-3′ (SEQ ID NO: 7570) KRAS-5345 Target:5′-TCTAAAATTTGTAATATTTTT-3′ (SEQ ID NO: 7571) KRAS-5349 Target:5′-AAATTTGTAATATTTTTGTCA-3′ (SEQ ID NO: 7572) KRAS-5350 Target:5′-AATTTGTAATATTTTTGTCAT-3′ (SEQ ID NO: 7573) KRAS-5351 Target:5′-ATTTGTAATATTTTTGTCATG-3′ (SEQ ID NO: 7574) KRAS-5355 Target:5′-GTAATATTTTTGTCATGAACT-3′ (SEQ ID NO: 7575) KRAS-5356 Target:5′-TAATATTTTTGTCATGAACTG-3′ (SEQ ID NO: 7576) KRAS-5372 Target:5′-AACTGTACTACTCCTAATTAT-3′ (SEQ ID NO: 7577) KRAS-5373 Target:5′-ACTGTACTACTCCTAATTATT-3′ (SEQ ID NO: 7578) KRAS-5383 Target:5′-TCCTAATTATTGTAATGTAAT-3′ (SEQ ID NO: 7579) KRAS-5384 Target:5′-CCTAATTATTGTAATGTAATA-3′ (SEQ ID NO: 7580) KRAS-5386 Target:5′-TAATTATTGTAATGTAATAAA-3′ (SEQ ID NO: 7581) KRAS-5388 Target:5′-ATTATTGTAATGTAATAAAAA-3′ (SEQ ID NO: 7582) KRAS-5389 Target:5′-TTATTGTAATGTAATAAAAAT-3′ (SEQ ID NO: 7583) KRAS-5391 Target:5′-ATTGTAATGTAATAAAAATAG-3′ (SEQ ID NO: 7584) KRAS-5392 Target:5′-TTGTAATGTAATAAAAATAGT-3′ (SEQ ID NO: 7585) KRAS-5396 Target:5′-AATGTAATAAAAATAGTTACA-3′ (SEQ ID NO: 7586) KRAS-5397 Target:5′-ATGTAATAAAAATAGTTACAG-3′ (SEQ ID NO: 7587) KRAS-5398 Target:5′-TGTAATAAAAATAGTTACAGT-3′ (SEQ ID NO: 7588) KRAS-5406 Target:5′-AAATAGTTACAGTGACAAAAA-3′ (SEQ ID NO: 7589) KRAS-5407 Target:5′-AATAGTTACAGTGACAAAAAA-3′ (SEQ ID NO: 7590) KRAS-5409 Target:5′-TAGTTACAGTGACAAAAAAAA-3′ (SEQ ID NO: 7591) KRAS-172 Target:5′-CCTGCTGAAAATGACTGAATA-3′ (SEQ ID NO: 7592) KRAS-178 Target:5′-GAAAATGACTGAATATAAACT-3′ (SEQ ID NO: 7593) KRAS-182 Target:5′-ATGACTGAATATAAACTTGTG-3′ (SEQ ID NO: 7594) KRAS-244 Target:5′-ACAGCTAATTCAGAATCATTT-3′ (SEQ ID NO: 7595) KRAS-268 Target:5′-GGACGAATATGATCCAACAAT-3′ (SEQ ID NO: 7596) KRAS-304 Target:5′-GAAGCAAGTAGTAATTGATGG-3′ (SEQ ID NO: 7597) KRAS-404 Target:5′-GGGGAGGGCTTTCTTTGTGTA-3′ (SEQ ID NO: 7598) KRAS-413 Target:5′-TTTCTTTGTGTATTTGCCATA-3′ (SEQ ID NO: 7599) KRAS-414 Target:5′-TTCTTTGTGTATTTGCCATAA-3′ (SEQ ID NO: 7600) KRAS-415 Target:5′-TCTTTGTGTATTTGCCATAAA-3′ (SEQ ID NO: 7601) KRAS-416 Target:5′-CTTTGTGTATTTGCCATAAAT-3′ (SEQ ID NO: 7602) KRAS-420 Target:5′-GTGTATTTGCCATAAATAATA-3′ (SEQ ID NO: 7603) KRAS-421 Target:5′-TGTATTTGCCATAAATAATAC-3′ (SEQ ID NO: 7604) KRAS-427 Target:5′-TGCCATAAATAATACTAAATC-3′ (SEQ ID NO: 7605) KRAS-428 Target:5′-GCCATAAATAATACTAAATCA-3′ (SEQ ID NO: 7606) KRAS-430 Target:5′-CATAAATAATACTAAATCATT-3′ (SEQ ID NO: 7607) KRAS-431 Target:5′-ATAAATAATACTAAATCATTT-3′ (SEQ ID NO: 7608) KRAS-432 Target:5′-TAAATAATACTAAATCATTTG-3′ (SEQ ID NO: 7609) KRAS-433 Target:5′-AAATAATACTAAATCATTTGA-3′ (SEQ ID NO: 7610) KRAS-446 Target:5′-TCATTTGAAGATATTCACCAT-3′ (SEQ ID NO: 7611) KRAS-448 Target:5′-ATTTGAAGATATTCACCATTA-3′ (SEQ ID NO: 7612) KRAS-449 Target:5′-TTTGAAGATATTCACCATTAT-3′ (SEQ ID NO: 7613) KRAS-450 Target:5′-TTGAAGATATTCACCATTATA-3′ (SEQ ID NO: 7614) KRAS-451 Target:5′-TGAAGATATTCACCATTATAG-3′ (SEQ ID NO: 7615) KRAS-461 Target:5′-CACCATTATAGAGAACAAATT-3′ (SEQ ID NO: 7616) KRAS-463 Target:5′-CCATTATAGAGAACAAATTAA-3′ (SEQ ID NO: 7617) KRAS-464 Target:5′-CATTATAGAGAACAAATTAAA-3′ (SEQ ID NO: 7618) KRAS-465 Target:5′-ATTATAGAGAACAAATTAAAA-3′ (SEQ ID NO: 7619) KRAS-466 Target:5′-TTATAGAGAACAAATTAAAAG-3′ (SEQ ID NO: 7620) KRAS-516 Target:5′-TCCTAGTAGGAAATAAATGTG-3′ (SEQ ID NO: 7621) KRAS-517 Target:5′-CCTAGTAGGAAATAAATGTGA-3′ (SEQ ID NO: 7622) KRAS-581 Target:5′-GCAAGAAGTTATGGAATTCCT-3′ (SEQ ID NO: 7623) KRAS-584 Target:5′-AGAAGTTATGGAATTCCTTTT-3′ (SEQ ID NO: 7624) KRAS-585 Target:5′-GAAGTTATGGAATTCCTTTTA-3′ (SEQ ID NO: 7625) KRAS-586 Target:5′-AAGTTATGGAATTCCTTTTAT-3′ (SEQ ID NO: 7626) KRAS-587 Target:5′-AGTTATGGAATTCCTTTTATT-3′ (SEQ ID NO: 7627) KRAS-634 Target:5′-AGTGGAGGATGCTTTTTATAC-3′ (SEQ ID NO: 7628) KRAS-636 Target:5′-TGGAGGATGCTTTTTATACAT-3′ (SEQ ID NO: 7629) KRAS-676 Target:5′-ATACAGATTGAAAAAAATCAG-3′ (SEQ ID NO: 7630) KRAS-677 Target:5′-TACAGATTGAAAAAAATCAGC-3′ (SEQ ID NO: 7631) KRAS-679 Target:5′-CAGATTGAAAAAAATCAGCAA-3′ (SEQ ID NO: 7632) KRAS-680 Target:5′-AGATTGAAAAAAATCAGCAAA-3′ (SEQ ID NO: 7633) KRAS-681 Target:5′-GATTGAAAAAAATCAGCAAAG-3′ (SEQ ID NO: 7634) KRAS-682 Target:5′-ATTGAAAAAAATCAGCAAAGA-3′ (SEQ ID NO: 7635) KRAS-710 Target:5′-ACTCCTGGCTGTGTGAAAATT-3′ (SEQ ID NO: 7636) KRAS-714 Target:5′-CTGGCTGTGTGAAAATTAAAA-3′ (SEQ ID NO: 7637) KRAS-718 Target:5′-CTGTGTGAAAATTAAAAAATG-3′ (SEQ ID NO: 7638) KRAS-719 Target:5′-TGTGTGAAAATTAAAAAATGC-3′ (SEQ ID NO: 7639) KRAS-720 Target:5′-GTGTGAAAATTAAAAAATGCA-3′ (SEQ ID NO: 7640) KRAS-721 Target:5′-TGTGAAAATTAAAAAATGCAT-3′ (SEQ ID NO: 7641) KRAS-723 Target:5′-TGAAAATTAAAAAATGCATTA-3′ (SEQ ID NO: 7642) KRAS-724 Target:5′-GAAAATTAAAAAATGCATTAT-3′ (SEQ ID NO: 7643) KRAS-725 Target:5′-AAAATTAAAAAATGCATTATA-3′ (SEQ ID NO: 7644) KRAS-726 Target:5′-AAATTAAAAAATGCATTATAA-3′ (SEQ ID NO: 7645) KRAS-728 Target:5′-ATTAAAAAATGCATTATAATG-3′ (SEQ ID NO: 7646) KRAS-735 Target:5′-AATGCATTATAATGTAATCTG-3′ (SEQ ID NO: 7647) KRAS-782 Target:5′-AGTTCGAGAAATTCGAAAACA-3′ (SEQ ID NO: 7648) KRAS-783 Target:5′-GTTCGAGAAATTCGAAAACAT-3′ (SEQ ID NO: 7649) KRAS-784 Target:5′-TTCGAGAAATTCGAAAACATA-3′ (SEQ ID NO: 7650) KRAS-787 Target:5′-GAGAAATTCGAAAACATAAAG-3′ (SEQ ID NO: 7651) KRAS-792 Target:5′-ATTCGAAAACATAAAGAAAAG-3′ (SEQ ID NO: 7652) KRAS-793 Target:5′-TTCGAAAACATAAAGAAAAGA-3′ (SEQ ID NO: 7653) KRAS-817 Target:5′-GCAAAGATGGTAAAAAGAAGA-3′ (SEQ ID NO: 7654) KRAS-818 Target:5′-CAAAGATGGTAAAAAGAAGAA-3′ (SEQ ID NO: 7655) KRAS-820 Target:5′-AAGATGGTAAAAAGAAGAAAA-3′ (SEQ ID NO: 7656) KRAS-821 Target:5′-AGATGGTAAAAAGAAGAAAAA-3′ (SEQ ID NO: 7657) KRAS-826 Target:5′-GTAAAAAGAAGAAAAAGAAGT-3′ (SEQ ID NO: 7658) KRAS-827 Target:5′-TAAAAAGAAGAAAAAGAAGTC-3′ (SEQ ID NO: 7659) KRAS-828 Target:5′-AAAAAGAAGAAAAAGAAGTCA-3′ (SEQ ID NO: 7660) KRAS-844 Target:5′-AGTCAAAGACAAAGTGTGTAA-3′ (SEQ ID NO: 7661) KRAS-845 Target:5′-GTCAAAGACAAAGTGTGTAAT-3′ (SEQ ID NO: 7662) KRAS-849 Target:5′-AAGACAAAGTGTGTAATTATG-3′ (SEQ ID NO: 7663) KRAS-851 Target:5′-GACAAAGTGTGTAATTATGTA-3′ (SEQ ID NO: 7664) KRAS-855 Target:5′-AAGTGTGTAATTATGTAAATA-3′ (SEQ ID NO: 7665) KRAS-857 Target:5′-GTGTGTAATTATGTAAATACA-3′ (SEQ ID NO: 7666) KRAS-859 Target:5′-GTGTAATTATGTAAATACAAT-3′ (SEQ ID NO: 7667) KRAS-860 Target:5′-TGTAATTATGTAAATACAATT-3′ (SEQ ID NO: 7668) KRAS-864 Target:5′-ATTATGTAAATACAATTTGTA-3′ (SEQ ID NO: 7669) KRAS-865 Target:5′-TTATGTAAATACAATTTGTAC-3′ (SEQ ID NO: 7670) KRAS-866 Target:5′-TATGTAAATACAATTTGTACT-3′ (SEQ ID NO: 7671) KRAS-867 Target:5′-ATGTAAATACAATTTGTACTT-3′ (SEQ ID NO: 7672) KRAS-873 Target:5′-ATACAATTTGTACTTTTTTCT-3′ (SEQ ID NO: 7673) KRAS-874 Target:5′-TACAATTTGTACTTTTTTCTT-3′ (SEQ ID NO: 7674) KRAS-899 Target:5′-CATACTAGTACAAGTGGTAAT-3′ (SEQ ID NO: 7675) KRAS-900 Target:5′-ATACTAGTACAAGTGGTAATT-3′ (SEQ ID NO: 7676) KRAS-906 Target:5′-GTACAAGTGGTAATTTTTGTA-3′ (SEQ ID NO: 7677) KRAS-907 Target:5′-TACAAGTGGTAATTTTTGTAC-3′ (SEQ ID NO: 7678) KRAS-914 Target:5′-GGTAATTTTTGTACATTACAC-3′ (SEQ ID NO: 7679) KRAS-915 Target:5′-GTAATTTTTGTACATTACACT-3′ (SEQ ID NO: 7680) KRAS-916 Target:5′-TAATTTTTGTACATTACACTA-3′ (SEQ ID NO: 7681) KRAS-917 Target:5′-AATTTTTGTACATTACACTAA-3′ (SEQ ID NO: 7682) KRAS-918 Target:5′-ATTTTTGTACATTACACTAAA-3′ (SEQ ID NO: 7683) KRAS-930 Target:5′-TACACTAAATTATTAGCATTT-3′ (SEQ ID NO: 7684) KRAS-932 Target:5′-CACTAAATTATTAGCATTTGT-3′ (SEQ ID NO: 7685) KRAS-933 Target:5′-ACTAAATTATTAGCATTTGTT-3′ (SEQ ID NO: 7686) KRAS-938 Target:5′-ATTATTAGCATTTGTTTTAGC-3′ (SEQ ID NO: 7687) KRAS-944 Target:5′-AGCATTTGTTTTAGCATTACC-3′ (SEQ ID NO: 7688) KRAS-945 Target:5′-GCATTTGTTTTAGCATTACCT-3′ (SEQ ID NO: 7689) KRAS-947 Target:5′-ATTTGTTTTAGCATTACCTAA-3′ (SEQ ID NO: 7690) KRAS-948 Target:5′-TTTGTTTTAGCATTACCTAAT-3′ (SEQ ID NO: 7691) KRAS-949 Target:5′-TTGTTTTAGCATTACCTAATT-3′ (SEQ ID NO: 7692) KRAS-950 Target:5′-TGTTTTAGCATTACCTAATTT-3′ (SEQ ID NO: 7693) KRAS-986 Target:5′-CAGACTGTTAGCTTTTACCTT-3′ (SEQ ID NO: 7694) KRAS-987 Target:5′-AGACTGTTAGCTTTTACCTTA-3′ (SEQ ID NO: 7695) KRAS-993 Target:5′-TTAGCTTTTACCTTAAATGCT-3′ (SEQ ID NO: 7696) KRAS-994 Target:5′-TAGCTTTTACCTTAAATGCTT-3′ (SEQ ID NO: 7697) KRAS-995 Target:5′-AGCTTTTACCTTAAATGCTTA-3′ (SEQ ID NO: 7698) KRAS-1001 Target:5′-TACCTTAAATGCTTATTTTAA-3′ (SEQ ID NO: 7699) KRAS-1002 Target:5′-ACCTTAAATGCTTATTTTAAA-3′ (SEQ ID NO: 7700) KRAS-1008 Target:5′-AATGCTTATTTTAAAATGACA-3′ (SEQ ID NO: 7701) KRAS-1010 Target:5′-TGCTTATTTTAAAATGACAGT-3′ (SEQ ID NO: 7702) KRAS-1027 Target:5′-CAGTGGAAGTTTTTTTTTCCT-3′ (SEQ ID NO: 7703) KRAS-1029 Target:5′-GTGGAAGTTTTTTTTTCCTCT-3′ (SEQ ID NO: 7704) KRAS-1030 Target:5′-TGGAAGTTTTTTTTTCCTCTA-3′ (SEQ ID NO: 7705) KRAS-1068 Target:5′-AGTTTTGGTTTTTGAACTAGC-3′ (SEQ ID NO: 7706) KRAS-1090 Target:5′-ATGCCTGTGAAAAAGAAACTG-3′ (SEQ ID NO: 7707) KRAS-1097 Target:5′-TGAAAAAGAAACTGAATACCT-3′ (SEQ ID NO: 7708) KRAS-1098 Target:5′-GAAAAAGAAACTGAATACCTA-3′ (SEQ ID NO: 7709) KRAS-1104 Target:5′-GAAACTGAATACCTAAGATTT-3′ (SEQ ID NO: 7710) KRAS-1145 Target:5′-CATGCAGTTGATTACTTCTTA-3′ (SEQ ID NO: 7711) KRAS-1146 Target:5′-ATGCAGTTGATTACTTCTTAT-3′ (SEQ ID NO: 7712) KRAS-1150 Target:5′-AGTTGATTACTTCTTATTTTT-3′ (SEQ ID NO: 7713) KRAS-1158 Target:5′-ACTTCTTATTTTTCTTACCAA-3′ (SEQ ID NO: 7714) KRAS-1159 Target:5′-CTTCTTATTTTTCTTACCAAT-3′ (SEQ ID NO: 7715) KRAS-1160 Target:5′-TTCTTATTTTTCTTACCAATT-3′ (SEQ ID NO: 7716) KRAS-1183 Target:5′-GAATGTTGGTGTGAAACAAAT-3′ (SEQ ID NO: 7717) KRAS-1186 Target:5′-TGTTGGTGTGAAACAAATTAA-3′ (SEQ ID NO: 7718) KRAS-1187 Target:5′-GTTGGTGTGAAACAAATTAAT-3′ (SEQ ID NO: 7719) KRAS-1195 Target:5′-GAAACAAATTAATGAAGCTTT-3′ (SEQ ID NO: 7720) KRAS-1196 Target:5′-AAACAAATTAATGAAGCTTTT-3′ (SEQ ID NO: 7721) KRAS-1228 Target:5′-ATTCTGTGTTTTATCTAGTCA-3′ (SEQ ID NO: 7722) KRAS-1229 Target:5′-TTCTGTGTTTTATCTAGTCAC-3′ (SEQ ID NO: 7723) KRAS-1232 Target:5′-TGTGTTTTATCTAGTCACATA-3′ (SEQ ID NO: 7724) KRAS-1233 Target:5′-GTGTTTTATCTAGTCACATAA-3′ (SEQ ID NO: 7725) KRAS-1238 Target:5′-TTATCTAGTCACATAAATGGA-3′ (SEQ ID NO: 7726) KRAS-1239 Target:5′-TATCTAGTCACATAAATGGAT-3′ (SEQ ID NO: 7727) KRAS-1247 Target:5′-CACATAAATGGATTAATTACT-3′ (SEQ ID NO: 7728) KRAS-1248 Target:5′-ACATAAATGGATTAATTACTA-3′ (SEQ ID NO: 7729) KRAS-1253 Target:5′-AATGGATTAATTACTAATTTC-3′ (SEQ ID NO: 7730) KRAS-1255 Target:5′-TGGATTAATTACTAATTTCAG-3′ (SEQ ID NO: 7731) KRAS-1256 Target:5′-GGATTAATTACTAATTTCAGT-3′ (SEQ ID NO: 7732) KRAS-1257 Target:5′-GATTAATTACTAATTTCAGTT-3′ (SEQ ID NO: 7733) KRAS-1285 Target:5′-TCTAATTGGTTTTTACTGAAA-3′ (SEQ ID NO: 7734) KRAS-1289 Target:5′-ATTGGTTTTTACTGAAACATT-3′ (SEQ ID NO: 7735) KRAS-1290 Target:5′-TTGGTTTTTACTGAAACATTG-3′ (SEQ ID NO: 7736) KRAS-1305 Target:5′-ACATTGAGGGAACACAAATTT-3′ (SEQ ID NO: 7737) KRAS-1306 Target:5′-CATTGAGGGAACACAAATTTA-3′ (SEQ ID NO: 7738) KRAS-1379 Target:5′-CCTGATGAATGTAAAGTTACA-3′ (SEQ ID NO: 7739) KRAS-1380 Target:5′-CTGATGAATGTAAAGTTACAC-3′ (SEQ ID NO: 7740) KRAS-1452 Target:5′-CCCCAAAATATTATATTTTTT-3′ (SEQ ID NO: 7741) KRAS-1453 Target:5′-CCCAAAATATTATATTTTTTC-3′ (SEQ ID NO: 7742) KRAS-1454 Target:5′-CCAAAATATTATATTTTTTCT-3′ (SEQ ID NO: 7743) KRAS-1455 Target:5′-CAAAATATTATATTTTTTCTA-3′ (SEQ ID NO: 7744) KRAS-1457 Target:5′-AAATATTATATTTTTTCTATA-3′ (SEQ ID NO: 7745) KRAS-1458 Target:5′-AATATTATATTTTTTCTATAA-3′ (SEQ ID NO: 7746) KRAS-1459 Target:5′-ATATTATATTTTTTCTATAAA-3′ (SEQ ID NO: 7747) KRAS-1460 Target:5′-TATTATATTTTTTCTATAAAA-3′ (SEQ ID NO: 7748) KRAS-1461 Target:5′-ATTATATTTTTTCTATAAAAA-3′ (SEQ ID NO: 7749) KRAS-1462 Target:5′-TTATATTTTTTCTATAAAAAG-3′ (SEQ ID NO: 7750) KRAS-1463 Target:5′-TATATTTTTTCTATAAAAAGA-3′ (SEQ ID NO: 7751) KRAS-1464 Target:5′-ATATTTTTTCTATAAAAAGAA-3′ (SEQ ID NO: 7752) KRAS-1469 Target:5′-TTTTCTATAAAAAGAAAAAAA-3′ (SEQ ID NO: 7753) KRAS-1470 Target:5′-TTTCTATAAAAAGAAAAAAAT-3′ (SEQ ID NO: 7754) KRAS-1472 Target:5′-TCTATAAAAAGAAAAAAATGG-3′ (SEQ ID NO: 7755) KRAS-1473 Target:5′-CTATAAAAAGAAAAAAATGGA-3′ (SEQ ID NO: 7756) KRAS-1474 Target:5′-TATAAAAAGAAAAAAATGGAA-3′ (SEQ ID NO: 7757) KRAS-1475 Target:5′-ATAAAAAGAAAAAAATGGAAA-3′ (SEQ ID NO: 7758) KRAS-1476 Target:5′-TAAAAAGAAAAAAATGGAAAA-3′ (SEQ ID NO: 7759) KRAS-1477 Target:5′-AAAAAGAAAAAAATGGAAAAA-3′ (SEQ ID NO: 7760) KRAS-1478 Target:5′-AAAAGAAAAAAATGGAAAAAA-3′ (SEQ ID NO: 7761) KRAS-1482 Target:5′-GAAAAAAATGGAAAAAAATTA-3′ (SEQ ID NO: 7762) KRAS-1483 Target:5′-AAAAAAATGGAAAAAAATTAC-3′ (SEQ ID NO: 7763) KRAS-1488 Target:5′-AATGGAAAAAAATTACAAGGC-3′ (SEQ ID NO: 7764) KRAS-1489 Target:5′-ATGGAAAAAAATTACAAGGCA-3′ (SEQ ID NO: 7765) KRAS-1490 Target:5′-TGGAAAAAAATTACAAGGCAA-3′ (SEQ ID NO: 7766) KRAS-1525 Target:5′-AGGCCATTTCCTTTTCACATT-3′ (SEQ ID NO: 7767) KRAS-1531 Target:5′-TTTCCTTTTCACATTAGATAA-3′ (SEQ ID NO: 7768) KRAS-1538 Target:5′-TTCACATTAGATAAATTACTA-3′ (SEQ ID NO: 7769) KRAS-1539 Target:5′-TCACATTAGATAAATTACTAT-3′ (SEQ ID NO: 7770) KRAS-1540 Target:5′-CACATTAGATAAATTACTATA-3′ (SEQ ID NO: 7771) KRAS-1595 Target:5′-CCAGTATGAAATGGGGATTAT-3′ (SEQ ID NO: 7772) KRAS-1604 Target:5′-AATGGGGATTATTATAGCAAC-3′ (SEQ ID NO: 7773) KRAS-1631 Target:5′-GGGGCTATATTTACATGCTAC-3′ (SEQ ID NO: 7774) KRAS-1632 Target:5′-GGGCTATATTTACATGCTACT-3′ (SEQ ID NO: 7775) KRAS-1633 Target:5′-GGCTATATTTACATGCTACTA-3′ (SEQ ID NO: 7776) KRAS-1634 Target:5′-GCTATATTTACATGCTACTAA-3′ (SEQ ID NO: 7777) KRAS-1635 Target:5′-CTATATTTACATGCTACTAAA-3′ (SEQ ID NO: 7778) KRAS-1640 Target:5′-TTTACATGCTACTAAATTTTT-3′ (SEQ ID NO: 7779) KRAS-1647 Target:5′-GCTACTAAATTTTTATAATAA-3′ (SEQ ID NO: 7780) KRAS-1648 Target:5′-CTACTAAATTTTTATAATAAT-3′ (SEQ ID NO: 7781) KRAS-1650 Target:5′-ACTAAATTTTTATAATAATTG-3′ (SEQ ID NO: 7782) KRAS-1651 Target:5′-CTAAATTTTTATAATAATTGA-3′ (SEQ ID NO: 7783) KRAS-1652 Target:5′-TAAATTTTTATAATAATTGAA-3′ (SEQ ID NO: 7784) KRAS-1653 Target:5′-AAATTTTTATAATAATTGAAA-3′ (SEQ ID NO:.7785) KRAS-1654 Target:5′-AATTTTTATAATAATTGAAAA-3′ (SEQ ID NO: 7786) KRAS-1655 Target:5′-ATTTTTATAATAATTGAAAAG-3′ (SEQ ID NO: 7787) KRAS-1656 Target:5′-TTTTTATAATAATTGAAAAGA-3′ (SEQ ID NO: 7788) KRAS-1657 Target:5′-TTTTATAATAATTGAAAAGAT-3′ (SEQ ID NO: 7789) KRAS-1658 Target:5′-TTTATAATAATTGAAAAGATT-3′ (SEQ ID NO: 7790) KRAS-1662 Target:5′-TAATAATTGAAAAGATTTTAA-3′ (SEQ ID NO: 7791) KRAS-1663 Target:5′-AATAATTGAAAAGATTTTAAC-3′ (SEQ ID NO: 7792) KRAS-1665 Target:5′-TAATTGAAAAGATTTTAACAA-3′ (SEQ ID NO: 7793) KRAS-1666 Target:5′-AATTGAAAAGATTTTAACAAG-3′ (SEQ ID NO: 7794) KRAS-1667 Target:5′-ATTGAAAAGATTTTAACAAGT-3′ (SEQ ID NO: 7795) KRAS-1668 Target:5′-TTGAAAAGATTTTAACAAGTA-3′ (SEQ ID NO: 7796) KRAS-1669 Target:5′-TGAAAAGATTTTAACAAGTAT-3′ (SEQ ID NO: 7797) KRAS-1670 Target:5′-GAAAAGATTTTAACAAGTATA-3′ (SEQ ID NO: 7798) KRAS-1671 Target:5′-AAAAGATTTTAACAAGTATAA-3′ (SEQ ID NO: 7799) KRAS-1672 Target:5′-AAAGATTTTAACAAGTATAAA-3′ (SEQ ID NO: 7800) KRAS-1673 Target:5′-AAGATTTTAACAAGTATAAAA-3′ (SEQ ID NO: 7801) KRAS-1680 Target:5′-TAACAAGTATAAAAAATTCTC-3′ (SEQ ID NO: 7802) KRAS-1681 Target:5′-AACAAGTATAAAAAATTCTCA-3′ (SEQ ID NO: 7803) KRAS-1682 Target:5′-ACAAGTATAAAAAATTCTCAT-3′ (SEQ ID NO: 7804) KRAS-1683 Target:5′-CAAGTATAAAAAATTCTCATA-3′ (SEQ ID NO: 7805) KRAS-1684 Target:5′-AAGTATAAAAAATTCTCATAG-3′ (SEQ ID NO: 7806) KRAS-1685 Target:5′-AGTATAAAAAATTCTCATAGG-3′ (SEQ ID NO: 7807) KRAS-1686 Target:5GTATAAAAAATTCTCATAGGA-3′ (SEQ ID NO: 7808) KRAS-1687 Target:5′-TATAAAAAATTCTCATAGGAA-3′ (SEQ ID NO: 7809) KRAS-1689 Target:5′-TAAAAAATTCTCATAGGAATT-3′ (SEQ ID NO: 7810) KRAS-1690 Target:5′-AAAAAATTCTCATAGGAATTA-3′ (SEQ ID NO: 7811) KRAS-1734 Target:5′-TGCTCTTTCATAGTATAACTT-3′ (SEQ ID NO: 7812) KRAS-1739 Target:5′-TTTCATAGTATAACTTTAAAT-3′ (SEQ ID NO: 7813) KRAS-1740 Target:5′-TTCATAGTATAACTTTAAATC-3′ (SEQ ID NO: 7814) KRAS-1751 Target:5′-ACTTTAAATCTTTTCTTCAAC-3′ (SEQ ID NO: 7815) KRAS-1752 Target:5′-CTTTAAATCTTTTCTTCAACT-3′ (SEQ ID NO: 7816) KRAS-1767 Target:5′-TCAACTTGAGTCTTTGAAGAT-3′ (SEQ ID NO: 7817) KRAS-1769 Target:5′-AACTTGAGTCTTTGAAGATAG-3′ (SEQ ID NO: 7818) KRAS-1770 Target:5′-ACTTGAGTCTTTGAAGATAGT-3′ (SEQ ID NO: 7819) KRAS-1781 Target:5′-TGAAGATAGTTTTAATTCTGC-3′ (SEQ ID NO: 7820) KRAS-1782 Target:5′-GAAGATAGTTTTAATTCTGCT-3′ (SEQ ID NO: 7821) KRAS-1783 Target:5′-AAGATAGTTTTAATTCTGCTT-3′ (SEQ ID NO: 7822) KRAS-1797 Target:5′-TCTGCTTGTGACATTAAAAGA-3′ (SEQ ID NO: 7823) KRAS-2045 Target:5′-AGAGCATTGCTTTTGTTTCTT-3′ (SEQ ID NO: 7824) KRAS-2046 Target:5′-GAGCATTGCTTTTGTTTCTTA-3′ (SEQ ID NO: 7825) KRAS-2052 Target:5′-TGCTTTTGTTTCTTAAGAAAA-3′ (SEQ ID NO: 7826) KRAS-2059 Target:5′-GTTTCTTAAGAAAACAAACTC-3′ (SEQ ID NO: 7827) KRAS-2060 Target:5′-TTTCTTAAGAAAACAAACTCT-3′ (SEQ ID NO: 7828) KRAS-2061 Target:5′-TTCTTAAGAAAACAAACTCTT-3′ (SEQ ID NO: 7829) KRAS-2062 Target:5′-TCTTAAGAAAACAAACTCTTT-3′ (SEQ ID NO: 7830) KRAS-2063 Target:5′-CTTAAGAAAACAAACTCTTTT-3′ (SEQ ID NO: 7831) KRAS-2064 Target:5′-TTAAGAAAACAAACTCTTTTT-3′ (SEQ ID NO: 7832) KRAS-2065 Target:5′-TAAGAAAACAAACTCTTTTTT-3′ (SEQ ID NO: 7833) KRAS-2069 Target:5′-AAAACAAACTCTTTTTTAAAA-3′ (SEQ ID NO: 7834) KRAS-2075 Target:5′-AACTCTTTTTTAAAAATTACT-3′ (SEQ ID NO: 7835) KRAS-2076 Target:5′-ACTCTTTTTTAAAAATTACTT-3′ (SEQ ID NO: 7836) KRAS-2077 Target:5′-CTCTTTTTTAAAAATTACTTT-3′ (SEQ ID NO: 7837) KRAS-2078 Target:5′-TCTTTTTTAAAAATTACTTTT-3′ (SEQ ID NO: 7838) KRAS-2079 Target:5′-CTTTTTTAAAAATTACTTTTA-3′ (SEQ ID NO: 7839) KRAS-2080 Target:5′-TTTTTTAAAAATTACTTTTAA-3′ (SEQ ID NO: 7840) KRAS-2081 Target:5′-TTTTTAAAAATTACTTTTAAA-3′ (SEQ ID NO: 7841) KRAS-2082 Target:5′-TTTTAAAAATTACTTTTAAAT-3′ (SEQ ID NO: 7842) KRAS-2083 Target:5′-TTTAAAAATTACTTTTAAATA-3′ (SEQ ID NO: 7843) KRAS-2090 Target:5′-ATTACTTTTAAATATTAACTC-3′ (SEQ ID NO: 7844) KRAS-2091 Target:5′-TTACTTTTAAATATTAACTCA-3′ (SEQ ID NO: 7845) KRAS-2093 Target:5′-ACTTTTAAATATTAACTCAAA-3′ (SEQ ID NO: 7846) KRAS-2095 Target:5′-TTTTAAATATTAACTCAAAAG-3′ (SEQ ID NO: 7847) KRAS-2096 Target:5′-TTTAAATATTAACTCAAAAGT-3′ (SEQ ID NO: 7848) KRAS-2097 Target:5′-TTAAATATTAACTCAAAAGTT-3′ (SEQ ID NO: 7849) KRAS-2098 Target:5′-TAAATATTAACTCAAAAGTTG-3′ (SEQ ID NO: 7850) KRAS-2132 Target:5′-GTGGTGTGCCAAGACATTAAT-3′ (SEQ ID NO: 7851) KRAS-2137 Target:5′-GTGCCAAGACATTAATTTTTT-3′ (SEQ ID NO: 7852) KRAS-2138 Target:5′-TGCCAAGACATTAATTTTTTT-3′ (SEQ ID NO: 7853) KRAS-2144 Target:5′-GACATTAATTTTTTTTTTAAA-3′ (SEQ ID NO: 7854) KRAS-2145 Target:5′-ACATTAATTTTTTTTTTAAAC-3′ (SEQ ID NO: 7855) KRAS-2147 Target:5′-ATTAATTTTTTTTTTAAACAA-3′ (SEQ ID NO: 7856) KRAS-2149 Target:5′-TAATTTTTTTTTTAAACAATG-3′ (SEQ ID NO: 7857) KRAS-2150 Target:5′-AATTTTTTTTTTAAACAATGA-3′ (SEQ ID NO: 7858) KRAS-2151 Target:5′-ATTTTTTTTTTAAACAATGAA-3′ (SEQ ID NO: 7859) KRAS-2152 Target:5′-TTTTTTTTTTAAACAATGAAG-3′ (SEQ ID NO: 7860) KRAS-2153 Target:5′-TTTTTTTTTAAACAATGAAGT-3′ (SEQ ID NO: 7861) KRAS-2155 Target:5′-TTTTTTTAAACAATGAAGTGA-3′ (SEQ ID NO: 7862) KRAS-2156 Target:5′-TTTTTTAAACAATGAAGTGAA-3′ (SEQ ID NO: 7863) KRAS-2157 Target:5′-TTTTTAAACAATGAAGTGAAA-3′ (SEQ ID NO: 7864) KRAS-2165 Target:5′-CAATGAAGTGAAAAAGTTTTA-3′ (SEQ ID NO: 7865) KRAS-2171 Target:5′-AGTGAAAAAGTTTTACAATCT-3′ (SEQ ID NO: 7866) KRAS-2172 Target:5′-GTGAAAAAGTTTTACAATCTC-3′ (SEQ ID NO: 7867) KRAS-2173 Target:5′-TGAAAAAGTTTTACAATCTCT-3′ (SEQ ID NO: 7868) KRAS-2214 Target:5′-ACACTGGTTAAATTAACATTG-3′ (SEQ ID NO: 7869) KRAS-2215 Target:5′-CACTGGTTAAATTAACATTGC-3′ (SEQ ID NO: 7870) KRAS-2216 Target:5′-ACTGGTTAAATTAACATTGCA-3′ (SEQ ID NO: 7871) KRAS-2227 Target:5′-TAACATTGCATAAACACTTTT-3′ (SEQ ID NO: 7872) KRAS-2245 Target:5′-TTTCAAGTCTGATCCATATTT-3′ (SEQ ID NO: 7873) KRAS-2255 Target:5′-GATCCATATTTAATAATGCTT-3′ (SEQ ID NO: 7874) KRAS-2256 Target:5′-ATCCATATTTAATAATGCTTT-3′ (SEQ ID NO: 7875) KRAS-2257 Target:5′-TCCATATTTAATAATGCTTTA-3′ (SEQ ID NO: 7876) KRAS-2258 Target:5′-CCATATTTAATAATGCTTTAA-3′ (SEQ ID NO: 7877) KRAS-2259 Target:5′-CATATTTAATAATGCTTTAAA-3′ (SEQ ID NO: 7878) KRAS-2260 Target:5′-ATATTTAATAATGCTTTAAAA-3′ (SEQ ID NO: 7879) KRAS-2262 Target:5′-ATTTAATAATGCTTTAAAATA-3′ (SEQ ID NO: 7880) KRAS-2263 Target:5′-TTTAATAATGCTTTAAAATAA-3′ (SEQ ID NO: 7881) KRAS-2264 Target:5′-TTAATAATGCTTTAAAATAAA-3′ (SEQ ID NO: 7882) KRAS-2265 Target:5′-TAATAATGCTTTAAAATAAAA-3′ (SEQ ID NO: 7883) KRAS-2272 Target:5′-GCTTTAAAATAAAAATAAAAA-3′ (SEQ ID NO: 7884) KRAS-2278 Target:5′-AAATAAAAATAAAAACAATCC-3′ (SEQ ID NO: 7885) KRAS-2279 Target:5′-AATAAAAATAAAAACAATCCT-3′ (SEQ ID NO: 7886) KRAS-2280 Target:5′-ATAAAAATAAAAACAATCCTT-3′ (SEQ ID NO: 7887) KRAS-2281 Target:5′-TAAAAATAAAAACAATCCTTT-3′ (SEQ ID NO: 7888) KRAS-2282 Target:5′-AAAAATAAAAACAATCCTTTT-3′ (SEQ ID NO: 7889) KRAS-2283 Target:5′-AAAATAAAAACAATCCTTTTG-3′ (SEQ ID NO: 7890) KRAS-2284 Target:5′-AAATAAAAACAATCCTTTTGA-3′ (SEQ ID NO: 7891) KRAS-2285 Target:5′-AATAAAAACAATCCTTTTGAT-3′ (SEQ ID NO: 7892) KRAS-2289 Target:5′-AAAACAATCCTTTTGATAAAT-3′ (SEQ ID NO: 7893) KRAS-2294 Target:5′-AATCCTTTTGATAAATTTAAA-3′ (SEQ ID NO: 7894) KRAS-2295 Target:5′-ATCCTTTTGATAAATTTAAAA-3′ (SEQ ID NO: 7895) KRAS-2296 Target:5′-TCCTTTTGATAAATTTAAAAT-3′ (SEQ ID NO: 7896) KRAS-2300 Target:5′-TTTGATAAATTTAAAATGTTA-3′ (SEQ ID NO: 7897) KRAS-2301 Target:5′-TTGATAAATTTAAAATGTTAC-3′ (SEQ ID NO: 7898) KRAS-2302 Target:5′-TGATAAATTTAAAATGTTACT-3′ (SEQ ID NO: 7899) KRAS-2303 Target:5′-GATAAATTTAAAATGTTACTT-3′ (SEQ ID NO: 7900) KRAS-2304 Target:5′-ATAAATTTAAAATGTTACTTA-3′ (SEQ ID NO: 7901) KRAS-2305 Target:5′-TAAATTTAAAATGTTACTTAT-3′ (SEQ ID NO: 7902) KRAS-2307 Target:5′-AATTTAAAATGTTACTTATTT-3′ (SEQ ID NO: 7903) KRAS-2308 Target:5′-ATTTAAAATGTTACTTATTTT-3′ (SEQ ID NO: 7904) KRAS-2309 Target:5′-TTTAAAATGTTACTTATTTTA-3′ (SEQ ID NO: 7905) KRAS-2310 Target:5′-TTAAAATGTTACTTATTTTAA-3′ (SEQ ID NO: 7906) KRAS-2311 Target:5′-TAAAATGTTACTTATTTTAAA-3′ (SEQ ID NO: 7907) KRAS-2313 Target:5′-AAATGTTACTTATTTTAAAAT-3′ (SEQ ID NO: 7908) KRAS-2318 Target:5′-TTACTTATTTTAAAATAAATG-3′ (SEQ ID NO: 7909) KRAS-2320 Target:5′-ACTTATTTTAAAATAAATGAA-3′ (SEQ ID NO: 7910) KRAS-2321 Target:5′-CTTATTTTAAAATAAATGAAG-3′ (SEQ ID NO: 7911) KRAS-2324 Target:5′-ATTTTAAAATAAATGAAGTGA-3′ (SEQ ID NO: 7912) KRAS-2325 Target:5′-TTTTAAAATAAATGAAGTGAG-3′ (SEQ ID NO: 7913) KRAS-2445 Target:5′-TATCCATTTCTTCATGTTAAA-3′ (SEQ ID NO: 7914) KRAS-2446 Target:5′-ATCCATTTCTTCATGTTAAAA-3′ (SEQ ID NO: 7915) KRAS-2473 Target:5′-ATCTCAAACTCTTAGTTTTTT-3′ (SEQ ID NO: 7916) KRAS-2483 Target:5′-CTTAGTTTTTTTTTTTTACAA-3′ (SEQ ID NO: 7917) KRAS-2484 Target:5′-TTAGTTTTTTTTTTTTACAAC-3′ (SEQ ID NO: 7918) KRAS-2485 Target:5′-TAGTTTTTTTTTTTTACAACT-3′ (SEQ ID NO: 7919) KRAS-2486 Target:5′-AGTTTTTTTTTTTTACAACTA-3′ (SEQ ID NO: 7920) KRAS-2487 Target:5′-GTTTTTTTTTTTTACAACTAT-3′ (SEQ ID NO: 7921) KRAS-2488 Target:5′-TTTTTTTTTTTTACAACTATG-3′ (SEQ ID NO: 7922) KRAS-2489 Target:5′-TTTTTTTTTTTACAACTATGT-3′ (SEQ ID NO: 7923) KRAS-2490 Target:5′-TTTTTTTTTTACAACTATGTA-3′ (SEQ ID NO: 7924) KRAS-2491 Target:5′-TTTTTTTTTACAACTATGTAA-3′ (SEQ ID NO: 7925) KRAS-2492 Target:5′-TTTTTTTTACAACTATGTAAT-3′ (SEQ ID NO: 7926) KRAS-2493 Target:5′-TTTTTTTACAACTATGTAATT-3′ (SEQ ID NO: 7927) KRAS-2500 Target:5′-ACAACTATGTAATTTATATTC-3′ (SEQ ID NO: 7928) KRAS-2501 Target:5′-CAACTATGTAATTTATATTCC-3′ (SEQ ID NO: 7929) KRAS-2502 Target:5′-AACTATGTAATTTATATTCCA-3′ (SEQ ID NO: 7930) KRAS-2508 Target:5′-GTAATTTATATTCCATTTACA-3′ (SEQ ID NO: 7931) KRAS-2509 Target:5′-TAATTTATATTCCATTTACAT-3′ (SEQ ID NO: 7932) KRAS-2510 Target:5′-AATTTATATTCCATTTACATA-3′ (SEQ ID NO: 7933) KRAS-2511 Target:5′-ATTTATATTCCATTTACATAA-3′ (SEQ ID NO: 7934) KRAS-2523 Target:5′-TTTACATAAGGATACACTTAT-3′ (SEQ ID NO: 7935) KRAS-2527 Target:5′-CATAAGGATACACTTATTTGT-3′ (SEQ ID NO: 7936) KRAS-2559 Target:5′-CAATCTGTAAATTTTTAACCT-3′ (SEQ ID NO: 7937) KRAS-2560 Target:5′-AATCTGTAAATTTTTAACCTA-3′ (SEQ ID NO: 7938) KRAS-2561 Target:5′-ATCTGTAAATTTTTAACCTAT-3′ (SEQ ID NO: 7939) KRAS-2617 Target:5′-TGTGCAAGAGGTGAAGTTTAT-3′ (SEQ ID NO: 7940) KRAS-2619 Target:5′-TGCAAGAGGTGAAGTTTATAT-3′ (SEQ ID NO: 7941) KRAS-2620 Target:5′-GCAAGAGGTGAAGTTTATATT-3′ (SEQ ID NO: 7942) KRAS-2622 Target:5′-AAGAGGTGAAGTTTATATTTG-3′ (SEQ ID NO: 7943) KRAS-2623 Target:5′-AGAGGTGAAGTTTATATTTGA-3′ (SEQ ID NO: 7944) KRAS-2628 Target:5′-TGAAGTTTATATTTGAATATC-3′ (SEQ ID NO: 7945) KRAS-2716 Target:5′-TGACTTGATGCAGTTTTAATA-3′ (SEQ ID NO: 7946) KRAS-2718 Target:5′-ACTTGATGCAGTTTTAATACT-3′ (SEQ ID NO: 7947) KRAS-2869 Target:5′-GGGATTTGACCTAATCACTAA-3′ (SEQ ID NO: 7948) KRAS-2875 Target:5′-TGACCTAATCACTAATTTTCA-3′ (SEQ ID NO: 7949) KRAS-2944 Target:5′-GACAGTAGGATTTTTCAAACC-3′ (SEQ ID NO: 7950) KRAS-2989 Target:5′-CCAGTGGAAGGAGAATTTAAT-3′ (SEQ ID NO: 7951) KRAS-2992 Target:5′-GTGGAAGGAGAATTTAATAAA-3′ (SEQ ID NO: 7952) KRAS-2994 Target:5′-GGAAGGAGAATTTAATAAAGA-3′ (SEQ ID NO: 7953) KRAS-2995 Target:5′-GAAGGAGAATTTAATAAAGAT-3′ (SEQ ID NO: 7954) KRAS-2999 Target:5′-GAGAATTTAATAAAGATAGTG-3′ (SEQ ID NO: 7955) KRAS-3028 Target:5′-ATTCCTTAGGTAATCTATAAC-3′ (SEQ ID NO: 7956) KRAS-3029 Target:5′-TTCCTTAGGTAATCTATAACT-3′ (SEQ ID NO: 7957) KRAS-3063 Target:5′-GTAACAGTAATACATTCCATT-3′ (SEQ ID NO: 7958) KRAS-3065 Target:5′-AACAGTAATACATTCCATTGT-3′ (SEQ ID NO: 7959) KRAS-3066 Target:5′-ACAGTAATACATTCCATTGTT-3′ (SEQ ID NO: 7960) KRAS-3067 Target:5′-CAGTAATACATTCCATTGTTT-3′ (SEQ ID NO: 7961) KRAS-3077 Target:5′-TTCCATTGTTTTAGTAACCAG-3′ (SEQ ID NO: 7962) KRAS-3091 Target:5′-TAACCAGAAATCTTCATGCAA-3′ (SEQ ID NO: 7963) KRAS-3101 Target:5′-TCTTCATGCAATGAAAAATAC-3′ (SEQ ID NO: 7964) KRAS-3108 Target:5′-GCAATGAAAAATACTTTAATT-3′ (SEQ ID NO: 7965) KRAS-3110 Target:5′-AATGAAAAATACTTTAATTCA-3′ (SEQ ID NO: 7966) KRAS-3111 Target:5′-ATGAAAAATACTTTAATTCAT-3′ (SEQ ID NO: 7967) KRAS-3125 Target:5′-AATTCATGAAGCTTACTTTTT-3′ (SEQ ID NO: 7968) KRAS-3128 Target:5′-TCATGAAGCTTACTTTTTTTT-3′ (SEQ ID NO: 7969) KRAS-3132 Target:5′-GAAGCTTACTTTTTTTTTTTG-3′ (SEQ ID NO: 7970) KRAS-3136 Target:5′-CTTACTTTTTTTTTTTGGTGT-3′ (SEQ ID NO: 7971) KRAS-3137 Target:5′-TTACTTTTTTTTTTTGGTGTC-3′ (SEQ ID NO: 7972) KRAS-3295 Target:5′-TCAACTAATTTTTGTATTTTT-3′ (SEQ ID NO: 7973) KRAS-3298 Target:5′-ACTAATTTTTGTATTTTTAGG-3′ (SEQ ID NO: 7974) KRAS-3411 Target:5′-AACTCATTTATTCAGCAAATA-3′ (SEQ ID NO: 7975) KRAS-3413 Target:5′-CTCATTTATTCAGCAAATATT-3′ (SEQ ID NO: 7976) KRAS-3415 Target:5′-CATTTATTCAGCAAATATTTA-3′ (SEQ ID NO: 7977) KRAS-3587 Target:5′-CGTATTTTAGTTTTGCAAAGA-3′ (SEQ ID NO: 7978) KRAS-3628 Target:5′-AGCTCTATAATTGTTTTGCTA-3′ (SEQ ID NO: 7979) KRAS-3675 Target:5′-GCTACTTTATGTAAATCACTT-3′ (SEQ ID NO: 7980) KRAS-3676 Target:5′-CTACTTTATGTAAATCACTTC-3′ (SEQ ID NO: 7981) KRAS-3677 Target:5′-TACTTTATGTAAATCACTTCA-3′ (SEQ ID NO: 7982) KRAS-3678 Target:5′-ACTTTATGTAAATCACTTCAT-3′ (SEQ ID NO: 7983) KRAS-3679 Target:5′-CTTTATGTAAATCACTTCATT-3′ (SEQ ID NO: 7984) KRAS-3680 Target:5′-TTTATGTAAATCACTTCATTG-3′ (SEQ ID NO: 7985) KRAS-3681 Target:5′-TTATGTAAATCACTTCATTGT-3′ (SEQ ID NO: 7986) KRAS-3695 Target:5′-TCATTGTTTTAAAGGAATAAA-3′ (SEQ ID NO: 7987) KRAS-3696 Target:5′-CATTGTTTTAAAGGAATAAAC-3′ (SEQ ID NO: 7988) KRAS-3699 Target:5′-TGTTTTAAAGGAATAAACTTG-3′ (SEQ ID NO: 7989) KRAS-3700 Target:5′-GTTTTAAAGGAATAAACTTGA-3′ (SEQ ID NO: 7990) KRAS-3701 Target:5′-TTTTAAAGGAATAAACTTGAT-3′ (SEQ ID NO: 7991) KRAS-3703 Target:5′-TTAAAGGAATAAACTTGATTA-3′ (SEQ ID NO: 7992) KRAS-3704 Target:5′-TAAAGGAATAAACTTGATTAT-3′ (SEQ ID NO: 7993) KRAS-3705 Target:5′-AAAGGAATAAACTTGATTATA-3′ (SEQ ID NO: 7994) KRAS-3706 Target:5′-AAGGAATAAACTTGATTATAT-3′ (SEQ ID NO: 7995) KRAS-3707 Target:5′-AGGAATAAACTTGATTATATT-3′ (SEQ ID NO: 7996) KRAS-3712 Target:5′-TAAACTTGATTATATTGTTTT-3′ (SEQ ID NO: 7997) KRAS-3713 Target:5′-AAACTTGATTATATTGTTTTT-3′ (SEQ ID NO: 7998) KRAS-3716 Target:5′-CTTGATTATATTGTTTTTTTA-3′ (SEQ ID NO: 7999) KRAS-3721 Target:5′-TTATATTGTTTTTTTATTTGG-3′ (SEQ ID NO: 8000) KRAS-3722 Target:5′-TATATTGTTTTTTTATTTGGC-3′ (SEQ ID NO: 8001) KRAS-3726 Target:5′-TTGTTTTTTTATTTGGCATAA-3′ (SEQ ID NO: 8002) KRAS-3727 Target:5′-TGTTTTTTTATTTGGCATAAC-3′ (SEQ ID NO: 8003) KRAS-3739 Target:5′-TGGCATAACTGTGATTCTTTT-3′ (SEQ ID NO: 8004) KRAS-3744 Target:5′-TAACTGTGATTCTTTTAGGAC-3′ (SEQ ID NO: 8005) KRAS-3745 Target:5′-AACTGTGATTCTTTTAGGACA-3′ (SEQ ID NO: 8006) KRAS-3781 Target:5′-AAGGTGTATGTCAGATATTCA-3′ (SEQ ID NO: 8007) KRAS-3782 Target:5′-AGGTGTATGTCAGATATTCAT-3′ (SEQ ID NO: 8008) KRAS-3808 Target:5′-CCCAAATGTGTAATATTCCAG-3′ (SEQ ID NO: 8009) KRAS-3836 Target:5′-TGCATAAGTAATTAAAATATA-3′ (SEQ ID NO: 8010) KRAS-3837 Target:5′-GCATAAGTAATTAAAATATAC-3′ (SEQ ID NO: 8011) KRAS-3838 Target:5′-CATAAGTAATTAAAATATACT-3′ (SEQ ID NO: 8012) KRAS-3839 Target:5′-ATAAGTAATTAAAATATACTT-3′ (SEQ ID NO: 8013) KRAS-3840 Target:5′-TAAGTAATTAAAATATACTTA-3′ (SEQ ID NO: 8014) KRAS-3841 Target:5′-AAGTAATTAAAATATACTTAA-3′ (SEQ ID NO: 8015) KRAS-3842 Target:5′-AGTAATTAAAATATACTTAAA-3′ (SEQ ID NO: 8016) KRAS-3843 Target:5′-GTAATTAAAATATACTTAAAA-3′ (SEQ ID NO: 8017) KRAS-3844 Target:5′-TAATTAAAATATACTTAAAAA-3′ (SEQ ID NO: 8018) KRAS-3846 Target:5′-ATTAAAATATACTTAAAAATT-3′ (SEQ ID NO: 8019) KRAS-3847 Target:5′-TTAAAATATACTTAAAAATTA-3′ (SEQ ID NO: 8020) KRAS-3848 Target:5′-TAAAATATACTTAAAAATTAA-3′ (SEQ ID NO: 8021) KRAS-3849 Target:5′-AAAATATACTTAAAAATTAAT-3′ (SEQ ID NO: 8022) KRAS-3853 Target:5′-TATACTTAAAAATTAATAGTT-3′ (SEQ ID NO: 8023) KRAS-3857 Target:5′-CTTAAAAATTAATAGTTTTAT-3′ (SEQ ID NO: 8024) KRAS-3858 Target:5′-TTAAAAATTAATAGTTTTATC-3′ (SEQ ID NO: 8025) KRAS-3859 Target:5′-TAAAAATTAATAGTTTTATCT-3′ (SEQ ID NO: 8026) KRAS-3874 Target:5′-TTATCTGGGTACAAATAAACA-3′ (SEQ ID NO: 8027) KRAS-3913 Target:5′-CAGACAAGGAAACTTCTATGT-3′ (SEQ ID NO: 8028) KRAS-3914 Target:5′-AGACAAGGAAACTTCTATGTA-3′ (SEQ ID NO: 8029) KRAS-3915 Target:5′-GACAAGGAAACTTCTATGTAA-3′ (SEQ ID NO: 8030) KRAS-3924 Target:5′-ACTTCTATGTAAAAATCACTA-3′ (SEQ ID NO: 8031) KRAS-3925 Target:5′-CTTCTATGTAAAAATCACTAT-3′ (SEQ ID NO: 8032) KRAS-3926 Target:5′-TTCTATGTAAAAATCACTATG-3′ (SEQ ID NO: 8033) KRAS-3930 Target:5′-ATGTAAAAATCACTATGATTT-3′ (SEQ ID NO: 8034) KRAS-3931 Target:5′-TGTAAAAATCACTATGATTTC-3′ (SEQ ID NO: 8035) KRAS-3940 Target:5′-CACTATGATTTCTGAATTGCT-3′ (SEQ ID NO: 8036) KRAS-3941 Target:5′-ACTATGATTTCTGAATTGCTA-3′ (SEQ ID NO: 8037) KRAS-3958 Target:5′-GCTATGTGAAACTACAGATCT-3′ (SEQ ID NO: 8038) KRAS-3959 Target:5′-CTATGTGAAACTACAGATCTT-3′ (SEQ ID NO: 8039) KRAS-3995 Target:5′-GTAGGGTGTTAAGACTTACAC-3′ (SEQ ID NO: 8040) KRAS-4082 Target:5′-TATTTAGGCCTCTTGAATTTT-3′ (SEQ ID NO: 8041) KRAS-4090 Target:5′-CCTCTTGAATTTTTGATGTAG-3′ (SEQ ID NO: 8042) KRAS-4091 Target:5′-CTCTTGAATTTTTGATGTAGA-3′ (SEQ ID NO: 8043) KRAS-4106 Target:5′-TGTAGATGGGCATTTTTTTAA-3′ (SEQ ID NO: 8044) KRAS-4112 Target:5′-TGGGCATTTTTTTAAGGTAGT-3′ (SEQ ID NO: 8045) KRAS-4124 Target:5′-TAAGGTAGTGGTTAATTACCT-3′ (SEQ ID NO: 8046) KRAS-4126 Target:5′-AGGTAGTGGTTAATTACCTTT-3′ (SEQ ID NO: 8047) KRAS-4127 Target:5′-GGTAGTGGTTAATTACCTTTA-3′ (SEQ ID NO: 8048) KRAS-4128 Target:5′-GTAGTGGTTAATTACCTTTAT-3′ (SEQ ID NO: 8049) KRAS-4129 Target:5′-TAGTGGTTAATTACCTTTATG-3′ (SEQ ID NO: 8050) KRAS-4139 Target:5′-TTACCTTTATGTGAACTTTGA-3′ (SEQ ID NO: 8051) KRAS-4144 Target:5′-TTTATGTGAACTTTGAATGGT-3′ (SEQ ID NO: 8052) KRAS-4150 Target:5′-TGAACTTTGAATGGTTTAACA-3′ (SEQ ID NO: 8053) KRAS-4154 Target:5′-CTTTGAATGGTTTAACAAAAG-3′ (SEQ ID NO: 8054) KRAS-4155 Target:5′-TTTGAATGGTTTAACAAAAGA-3′ (SEQ ID NO: 8055) KRAS-4156 Target:5′-TTGAATGGTTTAACAAAAGAT-3′ (SEQ ID NO: 8056) KRAS-4157 Target:5′-TGAATGGTTTAACAAAAGATT-3′ (SEQ ID NO: 8057) KRAS-4158 Target:5′-GAATGGTTTAACAAAAGATTT-3′ (SEQ ID NO: 8058) KRAS-4160 Target:5′-ATGGTTTAACAAAAGATTTGT-3′ (SEQ ID NO: 8059) KRAS-4161 Target:5′-TGGTTTAACAAAAGATTTGTT-3′ (SEQ ID NO: 8060) KRAS-4165 Target:5′-TTAACAAAAGATTTGTTTTTG-3′ (SEQ ID NO: 8061) KRAS-4166 Target:5′-TAACAAAAGATTTGTTTTTGT-3′ (SEQ ID NO: 8062) KRAS-4167 Target:5′-AACAAAAGATTTGTTTTTGTA-3′ (SEQ ID NO: 8063) KRAS-4169 Target:5′-CAAAAGATTTGTTTTTGTAGA-3′ (SEQ ID NO: 8064) KRAS-4170 Target:5′-AAAAGATTTGTTTTTGTAGAG-3′ (SEQ ID NO: 8065) KRAS-4171 Target:5′-AAAGATTTGTTTTTGTAGAGA-3′ (SEQ ID NO: 8066) KRAS-4172 Target:5′-AAGATTTGTTTTTGTAGAGAT-3′ (SEQ ID NO: 8067) KRAS-4178 Target:5′-TGTTTTTGTAGAGATTTTAAA-3′ (SEQ ID NO: 8068) KRAS-4195 Target:5′-TAAAGGGGGAGAATTCTAGAA-3′ (SEQ ID NO: 8069) KRAS-4197 Target:5′-AAGGGGGAGAATTCTAGAAAT-3′ (SEQ ID NO: 8070) KRAS-4198 Target:5′-AGGGGGAGAATTCTAGAAATA-3′ (SEQ ID NO: 8071) KRAS-4199 Target:5′-GGGGGAGAATTCTAGAAATAA-3′ (SEQ ID NO: 8072) KRAS-4200 Target:5′-GGGGAGAATTCTAGAAATAAA-3′ (SEQ ID NO: 8073) KRAS-4201 Target:5′-GGGAGAATTCTAGAAATAAAT-3′ (SEQ ID NO: 8074) KRAS-4206 Target:5′-AATTCTAGAAATAAATGTTAC-3′ (SEQ ID NO: 8075) KRAS-4207 Target:5′-ATTCTAGAAATAAATGTTACC-3′ (SEQ ID NO: 8076) KRAS-4208 Target:5′-TTCTAGAAATAAATGTTACCT-3′ (SEQ ID NO: 8077) KRAS-4209 Target:5′-TCTAGAAATAAATGTTACCTA-3′ (SEQ ID NO: 8078) KRAS-4210 Target:5′-CTAGAAATAAATGTTACCTAA-3′ (SEQ ID NO: 8079) KRAS-4212 Target:5′-AGAAATAAATGTTACCTAATT-3′ (SEQ ID NO: 8080) KRAS-4223 Target:5′-TTACCTAATTATTACAGCCTT-3′ (SEQ ID NO: 8081) KRAS-4224 Target:5′-TACCTAATTATTACAGCCTTA-3′ (SEQ ID NO: 8082) KRAS-4238 Target:5′-AGCCTTAAAGACAAAAATCCT-3′ (SEQ ID NO: 8083) KRAS-4241 Target:5′-CTTAAAGACAAAAATCCTTGT-3′ (SEQ ID NO: 8084) KRAS-4253 Target:5′-AATCCTTGTTGAAGTTTTTTT-3′ (SEQ ID NO: 8085) KRAS-4254 Target:5′-ATCCTTGTTGAAGTTTTTTTA-3′ (SEQ ID NO: 8086) KRAS-4256 Target:5′-CCTTGTTGAAGTTTTTTTAAA-3′ (SEQ ID NO: 8087) KRAS-4257 Target:5′-CTTGTTGAAGTTTTTTTAAAA-3′ (SEQ ID NO: 8088) KRAS-4261 Target:5′-TTGAAGTTTTTTTAAAAAAAG-3′ (SEQ ID NO: 8089) KRAS-4262 Target:5′-TGAAGTTTTTTTAAAAAAAGC-3′ (SEQ ID NO: 8090) KRAS-4263 Target:5′-GAAGTTTTTTTAAAAAAAGCT-3′ (SEQ ID NO: 8091) KRAS-4264 Target:5′-AAGTTTTTTTAAAAAAAGCTA-3′ (SEQ ID NO: 8092) KRAS-4265 Target:5′-AGTTTTTTTAAAAAAAGCTAA-3′ (SEQ ID NO: 8093) KRAS-4269 Target:5′-TTTTTAAAAAAAGCTAAATTA-3′ (SEQ ID NO: 8094) KRAS-4271 Target:5′-TTTAAAAAAAGCTAAATTACA-3′ (SEQ ID NO: 8095) KRAS-4293 Target:5′-AGACTTAGGCATTAACATGTT-3′ (SEQ ID NO: 8096) KRAS-4294 Target:5′-GACTTAGGCATTAACATGTTT-3′ (SEQ ID NO: 8097) KRAS-4325 Target:5′-ATAGCAGACGTATATTGTATC-3′ (SEQ ID NO: 8098) KRAS-4329 Target:5′-CAGACGTATATTGTATCATTT-3′ (SEQ ID NO: 8099) KRAS-4331 Target:5′-GACGTATATTGTATCATTTGA-3′ (SEQ ID NO: 8100) KRAS-4334 Target:5′-GTATATTGTATCATTTGAGTG-3′ (SEQ ID NO: 8101) KRAS-4335 Target:5′-TATATTGTATCATTTGAGTGA-3′ (SEQ ID NO: 8102) KRAS-4336 Target:5′-ATATTGTATCATTTGAGTGAA-3′ (SEQ ID NO: 8103) KRAS-4402 Target:5′-GCATAGGAATTTAGAACCTAA-3′ (SEQ ID NO: 8104) KRAS-4403 Target:5′-CATAGGAATTTAGAACCTAAC-3′ (SEQ ID NO: 8105) KRAS-4407 Target:5′-GGAATTTAGAACCTAACTTTT-3′ (SEQ ID NO: 8106) KRAS-4408 Target:5′-GAATTTAGAACCTAACTTTTA-3′ (SEQ ID NO: 8107) KRAS-4409 Target:5′-AATTTAGAACCTAACTTTTAT-3′ (SEQ ID NO: 8108) KRAS-4410 Target:5′-ATTTAGAACCTAACTTTTATA-3′ (SEQ ID NO: 8109) KRAS-4422 Target:5′-ACTTTTATAGGTTATCAAAAC-3′ (SEQ ID NO: 8110) KRAS-4424 Target:5′-TTTTATAGGTTATCAAAACTG-3′ (SEQ ID NO: 8111) KRAS-4458 Target:5′-ACAATTTTGTCCTAATATATA-3′ (SEQ ID NO: 8112) KRAS-4459 Target:5′-CAATTTTGTCCTAATATATAC-3′ (SEQ ID NO: 8113) KRAS-4460 Target:5′-AATTTTGTCCTAATATATACA-3′ (SEQ ID NO: 8114) KRAS-4466 Target:5′-GTCCTAATATATACATAGAAA-3′ (SEQ ID NO: 8115) KRAS-4471 Target:5′-AATATATACATAGAAACTTTG-3′ (SEQ ID NO: 8116) KRAS-4514 Target:5′-TGCACAAGTTCATCTCATTTG-3′ (SEQ ID NO: 8117) KRAS-4525 Target:5′-ATCTCATTTGTATTCCATTGA-3′ (SEQ ID NO: 8118) KRAS-4526 Target:5′-TCTCATTTGTATTCCATTGAT-3′ (SEQ ID NO: 8119) KRAS-4527 Target:5′-CTCATTTGTATTCCATTGATT-3′ (SEQ ID NO: 8120) KRAS-4528 Target:5′-TCATTTGTATTCCATTGATTT-3′ (SEQ ID NO: 8121) KRAS-4529 Target:5′-CATTTGTATTCCATTGATTTT-3′ (SEQ ID NO: 8122) KRAS-4530 Target:5′-ATTTGTATTCCATTGATTTTT-3′ (SEQ ID NO: 8123) KRAS-4531 Target:5′-TTTGTATTCCATTGATTTTTT-3′ (SEQ ID NO: 8124) KRAS-4538 Target:5′-TCCATTGATTTTTTTTTTCTT-3′ (SEQ ID NO: 8125) KRAS-4539 Target:5′-CCATTGATTTTTTTTTTCTTC-3′ (SEQ ID NO: 8126) KRAS-4543 Target:5′-TGATTTTTTTTTTCTTCTAAA-3′ (SEQ ID NO: 8127) KRAS-4544 Target:5′-GATTTTTTTTTTCTTCTAAAC-3′ (SEQ ID NO: 8128) KRAS-4545 Target:5′-ATTTTTTTTTTCTTCTAAACA-3′ (SEQ ID NO: 8129) KRAS-4546 Target:5′-TTTTTTTTTTCTTCTAAACAT-3′ (SEQ ID NO: 8130) KRAS-4547 Target:5′-TTTTTTTTTCTTCTAAACATT-3′ (SEQ ID NO: 8131) KRAS-4560 Target:5′-TAAACATTTTTTCTTCAAACA-3′ (SEQ ID NO: 8132) KRAS-4561 Target:5′-AAACATTTTTTCTTCAAACAG-3′ (SEQ ID NO: 8133) KRAS-4562 Target:5′-AACATTTTTTCTTCAAACAGT-3′ (SEQ ID NO: 8134) KRAS-4563 Target:5′-ACATTTTTTCTTCAAACAGTA-3′ (SEQ ID NO: 8135) KRAS-4564 Target:5′-CATTTTTTCTTCAAACAGTAT-3′ (SEQ ID NO: 8136) KRAS-4571 Target:5′-TCTTCAAACAGTATATAACTT-3′ (SEQ ID NO: 8137) KRAS-4576 Target:5′-AAACAGTATATAACTTTTTTT-3′ (SEQ ID NO: 8138) KRAS-4577 Target:5′-AACAGTATATAACTTTTTTTA-3′ (SEQ ID NO: 8139) KRAS-4578 Target:5′-ACAGTATATAACTTTTTTTAG-3′ (SEQ ID NO: 8140) KRAS-4579 Target:5′-CAGTATATAACTTTTTTTAGG-3′ (SEQ ID NO: 8141) KRAS-4585 Target:5′-ATAACTTTTTTTAGGGGATTT-3′ (SEQ ID NO: 8142) KRAS-4586 Target:5′-TAACTTTTTTTAGGGGATTTT-3′ (SEQ ID NO: 8143) KRAS-4597 Target:5′-AGGGGATTTTTTTTTAGACAG-3′ (SEQ ID NO: 8144) KRAS-4598 Target:5′-GGGGATTTTTTTTTAGACAGC-3′ (SEQ ID NO: 8145) KRAS-4599 Target:5′-GGGATTTTTTTTTAGACAGCA-3′ (SEQ ID NO: 8146) KRAS-4627 Target:5′-TCTGAAGATTTCCATTTGTCA-3′ (SEQ ID NO: 8147) KRAS-4628 Target:5′-CTGAAGATTTCCATTTGTCAA-3′ (SEQ ID NO: 8148) KRAS-4629 Target:5′-TGAAGATTTCCATTTGTCAAA-3′ (SEQ ID NO: 8149) KRAS-4630 Target:5′-GAAGATTTCCATTTGTCAAAA-3′ (SEQ ID NO: 8150) KRAS-4635 Target:5′-TTTCCATTTGTCAAAAAGTAA-3′ (SEQ ID NO: 8151) KRAS-4636 Target:5′-TTCCATTTGTCAAAAAGTAAT-3′ (SEQ ID NO: 8152) KRAS-4637 Target:5′-TCCATTTGTCAAAAAGTAATG-3′ (SEQ ID NO: 8153) KRAS-4642 Target:5′-TTGTCAAAAAGTAATGATTTC-3′ (SEQ ID NO: 8154) KRAS-4643 Target:5′-TGTCAAAAAGTAATGATTTCT-3′ (SEQ ID NO: 8155) KRAS-4644 Target:5′-GTCAAAAAGTAATGATTTCTT-3′ (SEQ ID NO: 8156) KRAS-4645 Target:5′-TCAAAAAGTAATGATTTCTTG-3′ (SEQ ID NO: 8157) KRAS-4647 Target:5′-AAAAAGTAATGATTTCTTGAT-3′ (SEQ ID NO: 8158) KRAS-4649 Target:5′-AAAGTAATGATTTCTTGATAA-3′ (SEQ ID NO: 8159) KRAS-4650 Target:5′-AAGTAATGATTTCTTGATAAT-3′ (SEQ ID NO: 8160) KRAS-4652 Target:5′-GTAATGATTTCTTGATAATTG-3′ (SEQ ID NO: 8161) KRAS-4653 Target:5′-TAATGATTTCTTGATAATTGT-3′ (SEQ ID NO: 8162) KRAS-4658 Target:5′-ATTTCTTGATAATTGTGTAGT-3′ (SEQ ID NO: 8163) KRAS-4659 Target:5′-TTTCTTGATAATTGTGTAGTA-3′ (SEQ ID NO: 8164) KRAS-4660 Target:5′-TTCTTGATAATTGTGTAGTAA-3′ (SEQ ID NO: 8165) KRAS-4662 Target:5′-CTTGATAATTGTGTAGTAATG-3′ (SEQ ID NO: 8166) KRAS-4665 Target:5′-GATAATTGTGTAGTAATGTTT-3′ (SEQ ID NO: 8167) KRAS-4667 Target:5′-TAATTGTGTAGTAATGTTTTT-3′ (SEQ ID NO: 8168) KRAS-4668 Target:5′-AATTGTGTAGTAATGTTTTTT-3′ (SEQ ID NO: 8169) KRAS-4702 Target:5′-TACCTTAAAGCTGAATTTATA-3′ (SEQ ID NO: 8170) KRAS-4704 Target:5′-CCTTAAAGCTGAATTTATATT-3′ (SEQ ID NO: 8171) KRAS-4713 Target:5′-TGAATTTATATTTAGTAACTT-3′ (SEQ ID NO: 8172) KRAS-4714 Target:5′-GAATTTATATTTAGTAACTTC-3′ (SEQ ID NO: 8173) KRAS-4715 Target:5′-AATTTATATTTAGTAACTTCT-3′ (SEQ ID NO: 8174) KRAS-4720 Target:5′-ATATTTAGTAACTTCTGTGTT-3′ (SEQ ID NO: 8175) KRAS-4771 Target:5′-AAACTGAATAGCTGTCATAAA-3′ (SEQ ID NO: 8176) KRAS-4772 Target:5′-AACTGAATAGCTGTCATAAAA-3′ (SEQ ID NO: 8177) KRAS-4786 Target:5′-CATAAAATGAAACTTTCTTTC-3′ (SEQ ID NO: 8178) KRAS-4787 Target:5′-ATAAAATGAAACTTTCTTTCT-3′ (SEQ ID NO: 8179) KRAS-4788 Target:5′-TAAAATGAAACTTTCTTTCTA-3′ (SEQ ID NO: 8180) KRAS-4790 Target:5′-AAATGAAACTTTCTTTCTAAA-3′ (SEQ ID NO: 8181) KRAS-4794 Target:5′-GAAACTTTCTTTCTAAAGAAA-3′ (SEQ ID NO: 8182) KRAS-4819 Target:5′-CTCACATGAGTTCTTGAAGAA-3′ (SEQ ID NO: 8183) KRAS-4828 Target:5′-GTTCTTGAAGAATAGTCATAA-3′ (SEQ ID NO: 8184) KRAS-4829 Target:5′-TTCTTGAAGAATAGTCATAAC-3′ (SEQ ID NO: 8185) KRAS-4830 Target:5′-TCTTGAAGAATAGTCATAACT-3′ (SEQ ID NO: 8186) KRAS-4833 Target:5′-TGAAGAATAGTCATAACTAGA-3′ (SEQ ID NO: 8187) KRAS-4834 Target:5′-GAAGAATAGTCATAACTAGAT-3′ (SEQ ID NO: 8188) KRAS-4835 Target:5′-AAGAATAGTCATAACTAGATT-3′ (SEQ ID NO: 8189) KRAS-4836 Target:5′-AGAATAGTCATAACTAGATTA-3′ (SEQ ID NO: 8190) KRAS-4840 Target:5′-TAGTCATAACTAGATTAAGAT-3′ (SEQ ID NO: 8191) KRAS-4846 Target:5′-TAACTAGATTAAGATCTGTGT-3′ (SEQ ID NO: 8192 KRAS-4848 Target:5′-ACTAGATTAAGATCTGTGTTT-3′ (SEQ ID NO: 8193) KRAS-4849 Target:5′-CTAGATTAAGATCTGTGTTTT-3′ (SEQ ID NO: 8194) KRAS-4851 Target:5′-AGATTAAGATCTGTGTTTTAG-3′ (SEQ ID NO: 8195) KRAS-4858 Target:5′-GATCTGTGTTTTAGTTTAATA-3′ (SEQ ID NO: 8196) KRAS-4859 Target:5′-ATCTGTGTTTTAGTTTAATAG-3′ (SEQ ID NO: 8197) KRAS-4861 Target:5′-CTGTGTTTTAGTTTAATAGTT-3′ (SEQ ID NO: 8198) KRAS-4863 Target:5′-GTGTTTTAGTTTAATAGTTTG-3′ (SEQ ID NO: 8199) KRAS-4864 Target:5′-TGTTTTAGTTTAATAGTTTGA-3′ (SEQ ID NO: 8200) KRAS-4890 Target:5′-CTGTTTGGGATAATGATAGGT-3′ (SEQ ID NO: 8201) KRAS-4892 Target:5′-GTTTGGGATAATGATAGGTAA-3′ (SEQ ID NO: 8202) KRAS-4895 Target:5′-TGGGATAATGATAGGTAATTT-3′ (SEQ ID NO: 8203) KRAS-4896 Target:5′-GGGATAATGATAGGTAATTTA-3′ (SEQ ID NO: 8204) KRAS-4899 Target:5′-ATAATGATAGGTAATTTAGAT-3′ (SEQ ID NO: 8205) KRAS-4900 Target:5′-TAATGATAGGTAATTTAGATG-3′ (SEQ ID NO: 8206) KRAS-4905 Target:5′-ATAGGTAATTTAGATGAATTT-3′ (SEQ ID NO: 8207) KRAS-4907 Target:5′-AGGTAATTTAGATGAATTTAG-3′ (SEQ ID NO: 8208) KRAS-4914 Target:5′-TTAGATGAATTTAGGGGAAAA-3′ (SEQ ID NO: 8209) KRAS-4915 Target:5′-TAGATGAATTTAGGGGAAAAA-3′ (SEQ ID NO: 8210) KRAS-4926 Target:5′-AGGGGAAAAAAAAGTTATCTG-3′ (SEQ ID NO: 8211) KRAS-4927 Target:5′-GGGGAAAAAAAAGTTATCTGC-3′ (SEQ ID NO: 8212) KRAS-4932 Target:5′-AAAAAAAGTTATCTGCAGATA-3′ (SEQ ID NO: 8213) KRAS-5033 Target:5′-CTGTCTTGTGTTTTCATGTTG-3′ (SEQ ID NO: 8214) KRAS-5034 Target:5′-TGTCTTGTGTTTTCATGTTGA-3′ (SEQ ID NO: 8215) KRAS-5035 Target:5′-GTCTTGTGTTTTCATGTTGAA-3′ (SEQ ID NO: 8216) KRAS-5045 Target:5′-TTCATGTTGAAAATACTTTTG-3′ (SEQ ID NO: 8217) KRAS-5046 Target:5′-TCATGTTGAAAATACTTTTGC-3′ (SEQ ID NO: 8218) KRAS-5047 Target:5′-CATGTTGAAAATACTTTTGCA-3′ (SEQ ID NO: 8219) KRAS-5048 Target:5′-ATGTTGAAAATACTTTTGCAT-3′ (SEQ ID NO: 8220) KRAS-5058 Target:5′-TACTTTTGCATTTTTCCTTTG-3′ (SEQ ID NO: 8221) KRAS-5075 Target:5′-TTTGAGTGCCAATTTCTTACT-3′ (SEQ ID NO: 8222) KRAS-5080 Target:5′-GTGCCAATTTCTTACTAGTAC-3′ (SEQ ID NO: 8223) KRAS-5081 Target:5′-TGCCAATTTCTTACTAGTACT-3′ (SEQ ID NO: 8224) KRAS-5090 Target:5′-CTTACTAGTACTATTTCTTAA-3′ (SEQ ID NO: 8225) KRAS-5091 Target:5′-TTACTAGTACTATTTCTTAAT-3′ (SEQ ID NO: 8226) KRAS-5096 Target:5′-AGTACTATTTCTTAATGTAAC-3′ (SEQ ID NO: 8227) KRAS-5097 Target:5′-GTACTATTTCTTAATGTAACA-3′ (SEQ ID NO: 8228) KRAS-5098 Target:5′-TACTATTTCTTAATGTAACAT-3′ (SEQ ID NO: 8229) KRAS-5105 Target:5′-TCTTAATGTAACATGTTTACC-3′ (SEQ ID NO: 8230) KRAS-5122 Target:5′-TACCTGGAATGTATTTTAACT-3′ (SEQ ID NO: 8231) KRAS-5124 Target:5′-CCTGGAATGTATTTTAACTAT-3′ (SEQ ID NO: 8232) KRAS-5125 Target:5′-CTGGAATGTATTTTAACTATT-3′ (SEQ ID NO: 8233) KRAS-5126 Target:5′-TGGAATGTATTTTAACTATTT-3′ (SEQ ID NO: 8234) KRAS-5127 Target:5′-GGAATGTATTTTAACTATTTT-3′ (SEQ ID NO: 8235) KRAS-5129 Target:5′-AATGTATTTTAACTATTTTTG-3′ (SEQ ID NO: 8236) KRAS-5131 Target:5′-TGTATTTTAACTATTTTTGTA-3′ (SEQ ID NO: 8237) KRAS-5132 Target:5′-GTATTTTAACTATTTTTGTAT-3′ (SEQ ID NO: 8238) KRAS-5133 Target:5′-TATTTTAACTATTTTTGTATA-3′ (SEQ ID NO: 8239) KRAS-5140 Target:5′-ACTATTTTTGTATAGTGTAAA-3′ (SEQ ID NO: 8240) KRAS-5141 Target:5′-CTATTTTTGTATAGTGTAAAC-3′ (SEQ ID NO: 8241) KRAS-5142 Target:5′-TATTTTTGTATAGTGTAAACT-3′ (SEQ ID NO: 8242) KRAS-5157 Target:5′-TAAACTGAAACATGCACATTT-3′ (SEQ ID NO: 8243) KRAS-5163 Target:5′-GAAACATGCACATTTTGTACA-3′ (SEQ ID NO: 8244) KRAS-5216 Target:5′-GATCCAGTTGTTTTCCATCAT-3′ (SEQ ID NO: 8245) KRAS-5218 Target:5′-TCCAGTTGTTTTCCATCATTT-3′ (SEQ ID NO: 8246) KRAS-5255 Target:5′-GAATGTTGGTCATATCAAACA-3′ (SEQ ID NO: 8247) KRAS-5256 Target:5′-AATGTTGGTCATATCAAACAT-3′ (SEQ ID NO: 8248) KRAS-5257 Target:5′-ATGTTGGTCATATCAAACATT-3′ (SEQ ID NO: 8249) KRAS-5261 Target:5′-TGGTCATATCAAACATTAAAA-3′ (SEQ ID NO: 8250) KRAS-5277 Target:5′-TAAAAATGACCACTCTTTTAA-3′ (SEQ ID NO: 8251) KRAS-5278 Target:5′-AAAAATGACCACTCTTTTAAT-3′ (SEQ ID NO: 8252) KRAS-5287 Target:5′-CACTCTTTTAATTGAAATTAA-3′ (SEQ ID NO: 8253) KRAS-5288 Target:5′-ACTCTTTTAATTGAAATTAAC-3′ (SEQ ID NO: 8254) KRAS-5290 Target:5′-TCTTTTAATTGAAATTAACTT-3′ (SEQ ID NO: 8255) KRAS-5292 Target:5′-TTTTAATTGAAATTAACTTTT-3′ (SEQ ID NO: 8256) KRAS-5293 Target:5′-TTTAATTGAAATTAACTTTTA-3′ (SEQ ID NO: 8257) KRAS-5294 Target:5′-TTAATTGAAATTAACTTTTAA-3′ (SEQ ID NO: 8258) KRAS-5295 Target:5′-TAATTGAAATTAACTTTTAAA-3′ (SEQ ID NO: 8259) KRAS-5296 Target:5′-AATTGAAATTAACTTTTAAAT-3′ (SEQ ID.NO: 8260) KRAS-5297 Target:5′-ATTGAAATTAACTTTTAAATG-3′ (SEQ ID NO: 8261) KRAS-5300 Target:5′-GAAATTAACTTTTAAATGTTT-3′ (SEQ ID NO: 8262) KRAS-5304 Target:5′-TTAACTTTTAAATGTTTATAG-3′ (SEQ ID NO: 8263) KRAS-5308 Target:5′-CTTTTAAATGTTTATAGGAGT-3′ (SEQ ID NO: 8264) KRAS-5309 Target:5′-TTTTAAATGTTTATAGGAGTA-3′ (SEQ ID NO: 8265) KRAS-5331 Target:5′-GTGCTGTGAAGTGATCTAAAA-3′ (SEQ ID NO: 8266) KRAS-5333 Target:5′-GCTGTGAAGTGATCTAAAATT-3′ (SEQ ID NO: 8267) KRAS-5334 Target:5′-CTGTGAAGTGATCTAAAATTT-3′ (SEQ ID NO: 8268) KRAS-5335 Target:5′-TGTGAAGTGATCTAAAATTTG-3′ (SEQ ID NO: 8269) KRAS-5336 Target:5′-GTGAAGTGATCTAAAATTTGT-3′ (SEQ ID NO: 8270) KRAS-5337 Target:5′-TGAAGTGATCTAAAATTTGTA-3′ (SEQ ID NO: 8271) KRAS-5338 Target:5′-GAAGTGATCTAAAATTTGTAA-3′ (SEQ ID NO: 8272) KRAS-5342 Target:5′-TGATCTAAAATTTGTAATATT-3′ (SEQ ID NO: 8273) KRAS-5343 Target:5′-GATCTAAAATTTGTAATATTT-3′ (SEQ ID NO: 8274) KRAS-5347 Target:5′-TAAAATTTGTAATATTTTTGT-3′ (SEQ ID NO: 8275) KRAS-5348 Target:5′-AAAATTTGTAATATTTTTGTC-3′ (SEQ ID NO: 8276) KRAS-5349 Target:5′-AAATTTGTAATATTTTTGTCA-3′ (SEQ ID NO: 8277) KRAS-5353 Target:5′-TTGTAATATTTTTGTCATGAA-3′ (SEQ ID NO: 8278) KRAS-5354 Target:5′-TGTAATATTTTTGTCATGAAC-3′ (SEQ ID NO: 8279) KRAS-5370 Target:5′-TGAACTGTACTACTCCTAATT-3′ (SEQ ID NO: 8280) KRAS-5371 Target:5′-GAACTGTACTACTCCTAATTA-3′ (SEQ ID NO: 8281) KRAS-5381 Target:5′-ACTCCTAATTATTGTAATGTA-3′ (SEQ ID NO: 8282) KRAS-5382 Target:5′-CTCCTAATTATTGTAATGTAA-3′ (SEQ ID NO: 8283) KRAS-5384 Target:5′-CCTAATTATTGTAATGTAATA-3′ (SEQ ID NO: 8284) KRAS-5386 Target:5′-TAATTATTGTAATGTAATAAA-3′ (SEQ ID NO: 8285) KRAS-5387 Target:5′-AATTATTGTAATGTAATAAAA-3′ (SEQ ID NO: 8286) KRAS-5389 Target:5′-TTATTGTAATGTAATAAAAAT-3′ (SEQ ID NO: 8287) KRAS-5390 Target:5′-TATTGTAATGTAATAAAAATA-3′ (SEQ ID NO: 8288) KRAS-5394 Target:5′-GTAATGTAATAAAAATAGTTA-3′ (SEQ ID NO: 8289) KRAS-5395 Target:5′-TAATGTAATAAAAATAGTTAC-3′ (SEQ ID NO: 8290) KRAS-5396 Target:5′-AATGTAATAAAAATAGTTACA-3′ (SEQ ID NO: 8291) KRAS-5404 Target:5′-AAAAATAGTTACAGTGACAAA-3′ (SEQ ID NO: 8292) KRAS-5405 Target:5′-AAAATAGTTACAGTGACAAAA-3′ (SEQ ID NO: 8293) KRAS-5407 Target:5′-AATAGTTACAGTGACAAAAAA-3′ (SEQ ID NO: 8294) KRAS-166 Target:5′-GAGAGGCCTGCTGAAAATGAC-3′ (SEQ ID NO: 8295) KRAS-167 Target:5′-AGAGGCCTGCTGAAAATGACT-3′ (SEQ ID NO: 8296) KRAS-168 Target:5′-GAGGCCTGCTGAAAATGACTG-3′ (SEQ ID NO: 8297) KRAS-169 Target:5′-AGGCCTGCTGAAAATGACTGA-3′ (SEQ ID NO: 8298) KRAS-204 Target:5′-TAGTTGGAGCTGGTGGCGTAG-3′ (SEQ ID NO: 8299) KRAS-205 Target:5′-AGTTGGAGCTGGTGGCGTAGG-3′ (SEQ ID NO: 8300) KRAS-206 Target:5′-GTTGGAGCTGGTGGCGTAGGC-3′ (SEQ ID NO: 8301) KRAS-207 Target:5′-TTGGAGCTGGTGGCGTAGGCA-3′ (SEQ ID NO: 8302) KRAS-208 Target:5′-TGGAGCTGGTGGCGTAGGCAA-3′ (SEQ ID NO: 8303) KRAS-209 Target:5′-GGAGCTGGTGGCGTAGGCAAG-3′ (SEQ ID NO: 8304) KRAS-210 Target:5′-GAGCTGGTGGCGTAGGCAAGA-3′ (SEQ ID NO: 8305) KRAS-241 Target:5′-GATACAGCTAATTCAGAATCA-3′ (SEQ ID NO: 8306) KRAS-313 Target:5′-AGTAATTGATGGAGAAACCTG-3′ (SEQ ID NO: 8307) KRAS-314 Target:5′-GTAATTGATGGAGAAACCTGT-3′ (SEQ ID NO: 8308) KRAS-318 Target:5′-TTGATGGAGAAACCTGTCTCT-3′ (SEQ ID NO: 8309) KRAS-328 Target:5′-AACCTGTCTCTTGGATATTCT-3′ (SEQ ID NO: 8310) KRAS-330 Target:5′-CCTGTCTCTTGGATATTCTCG-3′ (SEQ ID NO: 8311) KRAS-331 Target:5′-CTGTCTCTTGGATATTCTCGA-3′ (SEQ ID NO: 8312) KRAS-332 Target:5′-TGTCTCTTGGATATTCTCGAC-3′ (SEQ ID NO: 8313) KRAS-333 Target:5′-GTCTCTTGGATATTCTCGACA-3′ (SEQ ID NO: 8314) KRAS-334 Target:5′-TCTCTTGGATATTCTCGACAC-3′ (SEQ ID NO: 8315) KRAS-335 Target:5′-CTCTTGGATATTCTCGACACA-3′ (SEQ ID NO: 8316) KRAS-336 Target:5′-TCTTGGATATTCTCGACACAG-3′ (SEQ ID NO: 8317) KRAS-352 Target:5′-CACAGCAGGTCAAGAGGAGTA-3′ (SEQ ID NO: 8318) KRAS-353 Target:5′-ACAGCAGGTCAAGAGGAGTAC-3′ (SEQ ID NO: 8319) KRAS-354 Target:5′-CAGCAGGTCAAGAGGAGTACA-3′ (SEQ ID NO: 8320) KRAS-364 Target:5′-AGAGGAGTACAGTGCAATGAG-3′ (SEQ ID NO: 8321) KRAS-365 Target:5′-GAGGAGTACAGTGCAATGAGG-3′ (SEQ ID NO: 8322) KRAS-366 Target:5′-AGGAGTACAGTGCAATGAGGG-3′ (SEQ ID NO: 8323) KRAS-367 Target:5′-GGAGTACAGTGCAATGAGGGA-3′ (SEQ ID NO: 8324) KRAS-368 Target:5′-GAGTACAGTGCAATGAGGGAC-3′ (SEQ ID NO: 8325) KRAS-369 Target:5′-AGTACAGTGCAATGAGGGACC-3′ (SEQ ID NO: 8326) KRAS-370 Target:5′-GTACAGTGCAATGAGGGACCA-3′ (SEQ ID NO: 8327) KRAS-371 Target:5′-TACAGTGCAATGAGGGACCAG-3′ (SEQ ID NO: 8328) KRAS-372 Target:5′-ACAGTGCAATGAGGGACCAGT-3′ (SEQ ID NO: 8329) KRAS-420 Target:5′-GTGTATTTGCCATAAATAATA-3′ (SEQ ID NO: 8330) KRAS-421 Target:5′-TGTATTTGCCATAAATAATAC-3′ (SEQ ID NO: 8331) KRAS-422 Target:5′-GTATTTGCCATAAATAATACT-3′ (SEQ ID NO: 8332) KRAS-423 Target:5′-TATTTGCCATAAATAATACTA-3′ (SEQ ID NO: 8333) KRAS-424 Target:5′-ATTTGCCATAAATAATACTAA-3′ (SEQ ID NO: 8334) KRAS-425 Target:5′-TTTGCCATAAATAATACTAAA-3′ (SEQ ID NO: 8335) KRAS-426 Target:5′-TTGCCATAAATAATACTAAAT-3′ (SEQ ID NO: 8336) KRAS-436 Target:5′-TAATACTAAATCATTTGAAGA-3′ (SEQ ID NO: 8337) KRAS-437 Target:5′-AATACTAAATCATTTGAAGAT-3′ (SEQ ID NO: 8338) KRAS-438 Target:5′-ATACTAAATCATTTGAAGATA-3′ (SEQ ID NO: 8339) KRAS-439 Target:5′-TACTAAATCATTTGAAGATAT-3′ (SEQ ID NO: 8340) KRAS-440 Target:5′-ACTAAATCATTTGAAGATATT-3′ (SEQ ID NO: 8341) KRAS-441 Target:5′-CTAAATCATTTGAAGATATTC-3′ (SEQ ID NO: 8342) KRAS-442 Target:5′-TAAATCATTTGAAGATATTCA-3′ (SEQ ID NO: 8343) KRAS-443 Target:5′-AAATCATTTGAAGATATTCAC-3′ (SEQ ID NO: 8344) KRAS-444 Target:5′-AATCATTTGAAGATATTCACC-3′ (SEQ ID NO: 8345) KRAS-454 Target:5′-AGATATTCACCATTATAGAGA-3′ (SEQ ID NO: 8346) KRAS-455 Target:5′-GATATTCACCATTATAGAGAA-3′ (SEQ ID NO: 8347) KRAS-456 Target:5′-ATATTCACCATTATAGAGAAC-3′ (SEQ ID NO: 8348) KRAS-457 Target:5′-TATTCACCATTATAGAGAACA-3′ (SEQ ID NO: 8349) KRAS-458 Target:5′-ATTCACCATTATAGAGAACAA-3′ (SEQ ID NO: 8350) KRAS-459 Target:5′-TTCACCATTATAGAGAACAAA-3′ (SEQ ID NO: 8351) KRAS-460 Target:5′-TCACCATTATAGAGAACAAAT-3′ (SEQ ID NO: 8352) KRAS-461 Target:5′-CACCATTATAGAGAACAAATT-3′ (SEQ ID NO: 8353) KRAS-462 Target:5′-ACCATTATAGAGAACAAATTA-3′ (SEQ ID NO: 8354) KRAS-508 Target:5′-ACCTATGGTCCTAGTAGGAAA-3′ (SEQ ID NO: 8355) KRAS-531 Target:5′-AATGTGATTTGCCTTCTAGAA-3′ (SEQ ID NO: 8356) KRAS-532 Target:5′-ATGTGATTTGCCTTCTAGAAC-3′ (SEQ ID NO: 8357) KRAS-534 Target:5′-GTGATTTGCCTTCTAGAACAG-3′ (SEQ ID NO: 8358) KRAS-586 Target:5′-AAGTTATGGAATTCCTTTTAT-3′ (SEQ ID NO: 8359) KRAS-587 Target:5′-AGTTATGGAATTCCTTTTATT-3′ (SEQ ID NO: 8360) KRAS-588 Target:5′-GTTATGGAATTCCTTTTATTG-3′ (SEQ ID NO: 8361) KRAS-763 Target:5′-ATGATGCCTTCTATACATTAG-3′ (SEQ ID NO: 8362) KRAS-764 Target:5′-TGATGCCTTCTATACATTAGT-3′ (SEQ ID NO: 8363) KRAS-784 Target:5′-TTCGAGAAATTCGAAAACATA-3′ (SEQ ID NO: 8364) KRAS-794 Target:5′-TCGAAAACATAAAGAAAAGAT-3′ (SEQ ID NO: 8365) KRAS-795 Target:5′-CGAAAACATAAAGAAAAGATG-3′ (SEQ ID NO: 8366) KRAS-796 Target:5′-GAAAACATAAAGAAAAGATGA-3′ (SEQ ID NO: 8367) KRAS-797 Target:5′-AAAACATAAAGAAAAGATGAG-3′ (SEQ ID NO: 8368) KRAS-798 Target:5′-AAACATAAAGAAAAGATGAGC-3′ (SEQ ID NO: 8369) KRAS-799 Target:5′-AACATAAAGAAAAGATGAGCA-3′ (SEQ ID NO: 8370) KRAS-800 Target:5′-ACATAAAGAAAAGATGAGCAA-3′ (SEQ ID NO: 8371) KRAS-801 Target:5′-CATAAAGAAAAGATGAGCAAA-3′ (SEQ ID NO: 8372) KRAS-802 Target:5′-ATAAAGAAAAGATGAGCAAAG-3′ (SEQ ID NO: 8373) KRAS-908 Target:5′-ACAAGTGGTAATTTTTGTACA-3′ (SEQ ID NO: 8374) KRAS-909 Target:5′-CAAGTGGTAATTTTTGTACAT-3′ (SEQ ID NO: 8375) KRAS-920 Target:5′-TTTTGTACATTACACTAAATT-3′ (SEQ ID NO: 8376) KRAS-921 Target:5′-TTTGTACATTACACTAAATTA-3′ (SEQ ID NO: 8377) KRAS-922 Target:5′-TTGTACATTACACTAAATTAT-3′ (SEQ ID NO: 8378) KRAS-923 Target:5′-TGTACATTACACTAAATTATT-3′ (SEQ ID NO: 8379) KRAS-924 Target:5′-GTACATTACACTAAATTATTA-3′ (SEQ ID NO: 8380) KRAS-925 Target:5′-TACATTACACTAAATTATTAG-3′ (SEQ ID NO: 8381) KRAS-926 Target:5′-ACATTACACTAAATTATTAGC-3′ (SEQ ID NO: 8382) KRAS-927 Target:5′-CATTACACTAAATTATTAGCA-3′ (SEQ ID NO: 8383) KRAS-928 Target:5′-ATTACACTAAATTATTAGCAT-3′ (SEQ ID NO: 8384) KRAS-938 Target:5′-ATTATTAGCATTTGTTTTAGC-3′ (SEQ ID NO: 8385) KRAS-939 Target:5′-TTATTAGCATTTGTTTTAGCA-3′ (SEQ ID NO: 8386) KRAS-940 Target:5′-TATTAGCATTTGTTTTAGCAT-3′ (SEQ ID NO: 8387) KRAS-941 Target:5′-ATTAGCATTTGTTTTAGCATT-3′ (SEQ ID NO: 8388) KRAS-942 Target:5′-TTAGCATTTGTTTTAGCATTA-3′ (SEQ ID NO: 8389) KRAS-943 Target:5′-TAGCATTTGTTTTAGCATTAC-3′ (SEQ ID NO: 8390) KRAS-944 Target:5′-AGCATTTGTTTTAGCATTACC-3′ (SEQ ID NO: 8391) KRAS-945 Target:5′-GCATTTGTTTTAGCATTACCT-3′ (SEQ ID NO: 8392) KRAS-946 Target:5′-CATTTGTTTTAGCATTACCTA-3′ (SEQ ID NO: 8393) KRAS-1010 Target:5′-TGCTTATTTTAAAATGACAGT-3′ (SEQ ID NO: 8394) KRAS-1012 Target:5′-CTTATTTTAAAATGACAGTGG-3′ (SEQ ID NO: 8395) KRAS-1045 Target:5′-CCTCTAAGTGCCAGTATTCCC-3′ (SEQ ID NO: 8396) KRAS-1197 Target:5′-AACAAATTAATGAAGCTTTTG-3′ (SEQ ID NO: 8397) KRAS-1198 Target:5′-ACAAATTAATGAAGCTTTTGA-3′ (SEQ ID NO: 8398) KRAS-1230 Target:5′-TCTGTGTTTTATCTAGTCACA-3′ (SEQ ID NO: 8399) KRAS-1231 Target:5′-CTGTGTTTTATCTAGTCACAT-3′ (SEQ ID NO: 8400) KRAS-1234 Target:5′-TGTTTTATCTAGTCACATAAA-3′ (SEQ ID NO: 8401) KRAS-1249 Target:5′-CATAAATGGATTAATTACTAA-3′ (SEQ ID NO: 8402) KRAS-1250 Target:5′-ATAAATGGATTAATTACTAAT-3′ (SEQ ID NO: 8403) KRAS-1287 Target:5′-TAATTGGTTTTTACTGAAACA-3′ (SEQ ID NO: 8404) KRAS-1527 Target:5′-GCCATTTCCTTTTCACATTAG-3′ (SEQ ID NO: 8405) KRAS-1533 Target:5′-TCCTTTTCACATTAGATAAAT-3′ (SEQ ID NO: 8406) KRAS-1540 Target:5′-CACATTAGATAAATTACTATA-3′ (SEQ ID NO: 8407) KRAS-1541 Target:5′-ACATTAGATAAATTACTATAA-3′ (SEQ ID NO: 8408) KRAS-1542 Target:5′-CATTAGATAAATTACTATAAA-3′ (SEQ ID NO: 8409) KRAS-1583 Target:5′-GTTAAGGCAGACCCAGTATGA-3′ (SEQ ID NO: 8410) KRAS-1584 Target:5′-TTAAGGCAGACCCAGTATGAA-3′ (SEQ ID NO: 8411) KRAS-1585 Target:5′-TAAGGCAGACCCAGTATGAAA-3′ (SEQ ID NO: 8412) KRAS-1586 Target:5′-AAGGCAGACCCAGTATGAAAT-3′ (SEQ ID NO: 8413) KRAS-1597 Target:5′-AGTATGAAATGGGGATTATTA-3′ (SEQ ID NO: 8414) KRAS-1606 Target:5′-TGGGGATTATTATAGCAACCA-3′ (SEQ ID NO: 8415) KRAS-1630 Target:5′-TGGGGCTATATTTACATGCTA-3′ (SEQ ID NO: 8416) KRAS-1631 Target:5′-GGGGCTATATTTACATGCTAC-3′ (SEQ ID NO: 8417) KRAS-1632 Target:5′-GGGCTATATTTACATGCTACT-3′ (SEQ ID NO: 8418) KRAS-1633 Target:5′-GGCTATATTTACATGCTACTA-3′ (SEQ ID NO: 8419) KRAS-1634 Target:5′-GCTATATTTACATGCTACTAA-3′ (SEQ ID NO: 8420) KRAS-1635 Target:5′-CTATATTTACATGCTACTAAA-3′ (SEQ ID NO: 8421) KRAS-1636 Target:5′-TATATTTACATGCTACTAAAT-3′ (SEQ ID NO: 8422) KRAS-1637 Target:5′-ATATTTACATGCTACTAAATT-3′ (SEQ ID NO: 8423) KRAS-1638 Target:5′-TATTTACATGCTACTAAATTT-3′ (SEQ ID NO: 8424) KRAS-1639 Target:5′-ATTTACATGCTACTAAATTTT-3′ (SEQ ID NO: 8425) KRAS-1640 Target:5′-TTTACATGCTACTAAATTTTT-3′ (SEQ ID NO: 8426) KRAS-1736 Target:5′-CTCTTTCATAGTATAACTTTA-3′ (SEQ ID NO: 8427) KRAS-1741 Target:5′-TCATAGTATAACTTTAAATCT-3′ (SEQ ID NO: 8428) KRAS-1742 Target:5′-CATAGTATAACTTTAAATCTT-3′ (SEQ ID NO: 8429) KRAS-1753 Target:5′-TTTAAATCTTTTCTTCAACTT-3′ (SEQ ID NO: 8430) KRAS-1754 Target:5′-TTAAATCTTTTCTTCAACTTG-3′ (SEQ ID NO: 8431) KRAS-1769 Target:5′-AACTTGAGTCTTTGAAGATAG-3′ (SEQ ID NO: 8432) KRAS-1771 Target:5′-CTTGAGTCTTTGAAGATAGTT-3′ (SEQ ID NO: 8433) KRAS-1772 Target:5′-TTGAGTCTTTGAAGATAGTTT-3′ (SEQ ID NO: 8434) KRAS-1783 Target:5′-AAGATAGTTTTAATTCTGCTT-3′ (SEQ ID NO: 8435) KRAS-1784 Target:5′-AGATAGTTTTAATTCTGCTTG-3′ (SEQ ID NO: 8436) KRAS-1785 Target:5′-GATAGTTTTAATTCTGCTTGT-3′ (SEQ ID NO: 8437) KRAS-1799 Target:5′-TGCTTGTGACATTAAAAGATT-3′ (SEQ ID NO: 8438) KRAS-2100 Target:5′-AATATTAACTCAAAAGTTGAG-3′ (SEQ ID NO: 8439) KRAS-2134 Target:5′-GGTGTGCCAAGACATTAATTT-3′ (SEQ ID NO: 8440) KRAS-2216 Target:5′-ACTGGTTAAATTAACATTGCA-3′ (SEQ ID NO: 8441) KRAS-2217 Target:5′-CTGGTTAAATTAACATTGCAT-3′ (SEQ ID NO: 8442) KRAS-2218 Target:5′-TGGTTAAATTAACATTGCATA-3′ (SEQ ID NO: 8443) KRAS-2229 Target:5′-ACATTGCATAAACACTTTTCA-3′ (SEQ ID NO: 8444) KRAS-2247 Target:5′-TCAAGTCTGATCCATATTTAA-3′ (SEQ ID NO: 8445) KRAS-2326 Target:5′-TTTAAAATAAATGAAGTGAGA-3′ (SEQ ID NO: 8446) KRAS-2327 Target:5′-TTAAAATAAATGAAGTGAGAT-3′ (SEQ ID NO: 8447) KRAS-2547 Target:5′-TCAAGCTCAGCACAATCTGTA-3′ (SEQ ID NO: 8448) KRAS-2548 Target:5′-CAAGCTCAGCACAATCTGTAA-3′ (SEQ ID NO: 8449) KRAS-3741 Target:5′-GCATAACTGTGATTCTTTTAG-3′ (SEQ ID NO: 8450) KRAS-3746 Target:5′-ACTGTGATTCTTTTAGGACAA-3′ (SEQ ID NO: 8451) KRAS-3747 Target:5′-CTGTGATTCTTTTAGGACAAT-3′ (SEQ ID NO: 8452) KRAS-3783 Target:5′-GGTGTATGTCAGATATTCATA-3′ (SEQ ID NO: 8453) KRAS-3784 Target:5′-GTGTATGTCAGATATTCATAT-3′ (SEQ ID NO: 8454) KRAS-3810 Target:5′-CAAATGTGTAATATTCCAGTT-3′ (SEQ ID NO: 8455) KRAS-4396 Target:5′-CACACTGCATAGGAATTTAGA-3′ (SEQ ID NO: 8456) KRAS-4447 Target:5′-GTCACCATTGCACAATTTTGT-3′ (SEQ ID NO: 8457) KRAS-4448 Target:5′-TCACCATTGCACAATTTTGTC-3′ (SEQ ID NO: 8458) KRAS-4449 Target:5′-CACCATTGCACAATTTTGTCC-3′ (SEQ ID NO: 8459) KRAS-4450 Target:5′-ACCATTGCACAATTTTGTCCT-3′ (SEQ ID NO: 8460) KRAS-4451 Target:5′-CCATTGCACAATTTTGTCCTA-3′ (SEQ ID NO: 8461) KRAS-4452 Target:5′-CATTGCACAATTTTGTCCTAA-3′ (SEQ ID NO: 8462) KRAS-4748 Target:5′-GATAGCATGAATTCTGCATTG-3′ (SEQ ID NO: 8463) KRAS-4749 Target:5′-ATAGCATGAATTCTGCATTGA-3′ (SEQ ID NO: 8464) KRAS-4878 Target:5′-AGTTTGAAGTGCCTGTTTGGG-3′ (SEQ ID NO: 8465) KRAS-4879 Target:5′-GTTTGAAGTGCCTGTTTGGGA-3′ (SEQ ID NO: 8466) KRAS-4880 Target:5′-TTTGAAGTGCCTGTTTGGGAT-3′ (SEQ ID NO: 8467) KRAS-5073 Target:5′-CCTTTGAGTGCCAATTTCTTA-3′ (SEQ ID NO: 8468) KRAS-5074 Target:5′-CTTTGAGTGCCAATTTCTTAC-3′ (SEQ ID NO: 8469) KRAS-5075 Target:5′-TTTGAGTGCCAATTTCTTACT-3′ (SEQ ID NO: 8470) KRAS-5076 Target:5′-TTGAGTGCCAATTTCTTACTA-3′ (SEQ ID NO: 8471) KRAS-5077 Target:5′-TGAGTGCCAATTTCTTACTAG-3′ (SEQ ID NO: 8472) KRAS-5078 Target:5′-GAGTGCCAATTTCTTACTAGT-3′ (SEQ ID NO: 8473) KRAS-5128 Target:5′-GAATGTATTTTAACTATTTTT-3′ (SEQ ID NO: 8474) KRAS-5129 Target:5′-AATGTATTTTAACTATTTTTG-3′ (SEQ ID NO: 8475) KRAS-5138 Target:5′-TAACTATTTTTGTATAGTGTA-3′ (SEQ ID NO: 8476) KRAS-5139 Target:5′-AACTATTTTTGTATAGTGTAA-3′ (SEQ ID NO: 8477) KRAS-5140 Target:5′-ACTATTTTTGTATAGTGTAAA-3′ (SEQ ID NO: 8478) KRAS-5141 Target:5′-CTATTTTTGTATAGTGTAAAC-3′ (SEQ ID NO: 8479) KRAS-5142 Target:5′-TATTTTTGTATAGTGTAAACT-3′ (SEQ ID NO: 8480) KRAS-5143 Target:5′-ATTTTTGTATAGTGTAAACTG-3′ (SEQ ID NO: 8481) KRAS-5163 Target:5′-GAAACATGCACATTTTGTACA-3′ (SEQ ID NO: 8482) KRAS-5164 Target:5′-AAACATGCACATTTTGTACAT-3′ (SEQ ID NO: 8483) KRAS-5167 Target:5′-CATGCACATTTTGTACATTGT-3′ (SEQ ID NO: 8484) KRAS-5168 Target:5′-ATGCACATTTTGTACATTGTG-3′ (SEQ ID NO: 8485) KRAS-5169 Target:5′-TGCACATTTTGTACATTGTGC-3′ (SEQ ID NO: 8486) KRAS-5170 Target:5′-GCACATTTTGTACATTGTGCT-3′ (SEQ ID NO: 8487) KRAS-5171 Target:5′-CACATTTTGTACATTGTGCTT-3′ (SEQ ID NO: 8488) KRAS-5172 Target:5′-ACATTTTGTACATTGTGCTTT-3′ (SEQ ID NO: 8489) KRAS-5173 Target:5′-CATTTTGTACATTGTGCTTTC-3′ (SEQ ID NO: 8490) KRAS-5197 Target:5′-TGTGGGACATATGCAGTGTGA-3′ (SEQ ID NO: 8491) KRAS-5198 Target:5′-GTGGGACATATGCAGTGTGAT-3′ (SEQ ID NO: 8492) KRAS-5199 Target:5′-TGGGACATATGCAGTGTGATC-3′ (SEQ ID NO: 8493) KRAS-5200 Target:5′-GGGACATATGCAGTGTGATCC-3′ (SEQ ID NO: 8494) KRAS-5201 Target:5′-GGACATATGCAGTGTGATCCA-3′ (SEQ ID NO: 8495) KRAS-5202 Target:5′-GACATATGCAGTGTGATCCAG-3′ (SEQ ID NO: 8496) KRAS-5203 Target:5′-ACATATGCAGTGTGATCCAGT-3′ (SEQ ID NO: 8497) KRAS-5204 Target:5′-CATATGCAGTGTGATCCAGTT-3′ (SEQ ID NO: 8498) KRAS-5205 Target:5′-ATATGCAGTGTGATCCAGTTG-3′ (SEQ ID NO: 8499) KRAS-5209 Target:5′-GCAGTGTGATCCAGTTGTTTT-3′ (SEQ ID NO: 8500) KRAS-5210 Target:5′-CAGTGTGATCCAGTTGTTTTC-3′ (SEQ ID NO: 8501) KRAS-5211 Target:5′-AGTGTGATCCAGTTGTTTTCC-3′ (SEQ ID NO: 8502) KRAS-5212 Target:5′-GTGTGATCCAGTTGTTTTCCA-3′ (SEQ ID NO: 8503) KRAS-5213 Target:5′-TGTGATCCAGTTGTTTTCCAT-3′ (SEQ ID NO: 8504) KRAS-5214 Target:5′-GTGATCCAGTTGTTTTCCATC-3′ (SEQ ID NO: 8505) KRAS-5234 Target:5′-CATTTGGTTGCGCTGACCTAG-3′ (SEQ ID NO: 8506) KRAS-5235 Target:5′-ATTTGGTTGCGCTGACCTAGG-3′ (SEQ ID NO: 8507) KRAS-5252 Target:5′-TAGGAATGTTGGTCATATCAA-3′ (SEQ ID NO: 8508) KRAS-5253 Target:5′-AGGAATGTTGGTCATATCAAA-3′ (SEQ ID NO: 8509) KRAS-5254 Target:5′-GGAATGTTGGTCATATCAAAC-3′ (SEQ ID NO: 8510) KRAS-5255 Target:5′-GAATGTTGGTCATATCAAACA-3′ (SEQ ID NO: 8511) KRAS-5256 Target:5′-AATGTTGGTCATATCAAACAT-3′ (SEQ ID NO: 8512) KRAS-5257 Target:5′-ATGTTGGTCATATCAAACATT-3′ (SEQ ID NO: 8513) KRAS-5258 Target:5′-TGTTGGTCATATCAAACATTA-3′ (SEQ ID NO: 8514) KRAS-5259 Target:5′-GTTGGTCATATCAAACATTAA-3′ (SEQ ID NO: 8515) KRAS-5260 Target:5′-TTGGTCATATCAAACATTAAA-3′ (SEQ ID NO: 8516) KRAS-5299 Target:5′-TGAAATTAACTTTTAAATGTT-3′ (SEQ ID NO: 8517) KRAS-5300 Target:5′-GAAATTAACTTTTAAATGTTT-3′ (SEQ ID NO: 8518) KRAS-5304 Target:5′-TTAACTTTTAAATGTTTATAG-3′ (SEQ ID NO: 8519) KRAS-5305 Target:5′-TAACTTTTAAATGTTTATAGG-3′ (SEQ ID NO: 8520) KRAS-5306 Target:5′-AACTTTTAAATGTTTATAGGA-3′ (SEQ ID NO: 8521) KRAS-5307 Target:5′-ACTTTTAAATGTTTATAGGAG-3′ (SEQ ID NO: 8522) KRAS-5308 Target:5′-CTTTTAAATGTTTATAGGAGT-3′ (SEQ ID NO: 8523) KRAS-5309 Target:5′-TTTTAAATGTTTATAGGAGTA-3′ (SEQ ID NO: 8524) KRAS-5347 Target:5′-TAAAATTTGTAATATTTTTGT-3′ (SEQ ID NO: 8525) KRAS-5348 Target:5′-AAAATTTGTAATATTTTTGTC-3′ (SEQ ID NO: 8526) KRAS-5349 Target:5′-AAATTTGTAATATTTTTGTCA-3′ (SEQ ID NO: 8527) KRAS-5350 Target:5′-AATTTGTAATATTTTTGTCAT-3′ (SEQ ID NO: 8528) KRAS-5351 Target:5′-ATTTGTAATATTTTTGTCATG-3′ (SEQ ID NO: 8529) KRAS-5352 Target:5′-TTTGTAATATTTTTGTCATGA-3′ (SEQ ID NO: 8530) KRAS-5353 Target:5′-TTGTAATATTTTTGTCATGAA-3′ (SEQ ID NO: 8531) KRAS-5354 Target:5′-TGTAATATTTTTGTCATGAAC-3′ (SEQ ID NO: 8532) KRAS-5389 Target:5′-TTATTGTAATGTAATAAAAAT-3′ (SEQ ID NO: 8533) KRAS-5390 Target:5′-TATTGTAATGTAATAAAAATA-3′ (SEQ ID NO: 8534) KRAS-5391 Target:5′-ATTGTAATGTAATAAAAATAG-3′ (SEQ ID NO: 8535) KRAS-5392 Target:5′-TTGTAATGTAATAAAAATAGT-3′ (SEQ ID NO: 8536) KRAS-5393 Target:5′-TGTAATGTAATAAAAATAGTT-3′ (SEQ ID NO: 8537)

Within Tables 2-7 above, underlined residues indicate 2′-O-methylresidues, UPPER CASE indicates ribonucleotides, lower case denotesdeoxyribonucleotides, and in Table 2, “P-” indicates a 5′-terminalphosphate group. The above DsiRNA agents of Tables 3-6 are 25/27meragents possessing a blunt end. The structures and/or modificationpatterning of the agents of Tables 3-6 can be readily adapted to theabove generic sequence structures, e.g., the 3′ overhang of the secondstrand can be extended or contracted, 2′-O-methylation of the secondstrand can be expanded towards the 5′ end of the second strand,optionally at alternating sites, etc. Such further modifications areoptional, as 25/27mer DsiRNAs with such modifications can also bereadily designed from the above DsiRNA agents and are also expected tobe functional inhibitors of KRAS expression.

In certain embodiments, the DsiRNA agents of the invention require,e.g., at least 19, at least 20, at least 21, at least 22, at least 23,at least 24, at least 25 or at least 26 residues of the first strand tobe complementary to corresponding residues of the second strand. Incertain related embodiments, these first strand residues complementaryto corresponding residues of the second strand are optionallyconsecutive residues.

As used herein “DsiRNAmm” refers to a DisRNA having a “mismatch tolerantregion” containing one, two, three or four mismatched base pairs of theduplex formed by the sense and antisense strands of the DsiRNA, wheresuch mismatches are positioned within the DsiRNA at a location(s) lyingbetween (and thus not including) the two terminal base pairs of eitherend of the DsiRNA. The mismatched base pairs are located within a“mismatch-tolerant region” which is defined herein with respect to thelocation of the projected Ago2 cut site of the corresponding targetnucleic acid. The mismatch tolerant region is located “upstream of” theprojected Ago2 cut site of the target strand. “Upstream” in this contextwill be understood as the 5′-most portion of the DsiRNAmm duplex, where5′ refers to the orientation of the sense strand of the DsiRNA duplex.Therefore, the mismatch tolerant region is upstream of the base on thesense (passenger) strand that corresponds to the projected Ago2 cut siteof the target nucleic acid (see FIG. 1); alternatively, when referringto the antisense (guide) strand of the DsiRNAmm, the mismatch tolerantregion can also be described as positioned downstream of the base thatis complementary to the projected Ago2 cut site of the target nucleicacid, that is, the 3′-most portion of the antisense strand of theDsiRNAmm (where position 1 of the antisense strand is the 5′ terminalnucleotide of the antisense strand, see FIG. 1).

In one embodiment, for example with numbering as depicted in FIG. 1, themismatch tolerant region is positioned between and including base pairs3-9 when numbered from the nucleotide starting at the 5′ end of thesense strand of the duplex. Therefore, a DsiRNAmm of the inventionpossesses a single mismatched base pair at any one of positions 3, 4, 5,6, 7, 8 or 9 of the sense strand of a right-hand extended DsiRNA (whereposition 1 is the 5′ terminal nucleotide of the sense strand andposition 9 is the nucleotide residue of the sense strand that isimmediately 5′ of the projected Ago2 cut site of the target KRAS RNAsequence corresponding to the sense strand sequence). In certainembodiments, for a DsiRNAmm that possesses a mismatched base pairnucleotide at any of positions 3, 4, 5, 6, 7, 8 or 9 of the sensestrand, the corresponding mismatched base pair nucleotide of theantisense strand not only forms a mismatched base pair with the DsiRNAmmsense strand sequence, but also forms a mismatched base pair with aDsiRNAmm target KRAS RNA sequence (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, and complementarity issimilarly disrupted between the antisense strand sequence of theDsiRNAmm and the target KRAS RNA sequence). In alternative embodiments,the mismatch base pair nucleotide of the antisense strand of a DsiRNAmmonly form a mismatched base pair with a corresponding nucleotide of thesense strand sequence of the DsiRNAmm, yet base pairs with itscorresponding target KRAS RNA sequence nucleotide (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, yetcomplementarity is maintained between the antisense strand sequence ofthe DsiRNAmm and the target KRAS RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region (mismatch region) as described above(e.g., a DsiRNAmm harboring a mismatched nucleotide residue at any oneof positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand) can furtherinclude one, two or even three additional mismatched base pairs. Inpreferred embodiments, these one, two or three additional mismatchedbase pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and/or9 of the sense strand (and at corresponding residues of the antisensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of the sensestrand can occur, e.g., at nucleotides of both position 4 and position 6of the sense strand (with mismatch also occurring at correspondingnucleotide residues of the antisense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that base pair with theantisense strand sequence (e.g., for a DsiRNAmm possessing mismatchednucleotides at positions 3 and 6, but not at positions 4 and 5, themismatched residues of sense strand positions 3 and 6 are interspersedby two nucleotides that form matched base pairs with correspondingresidues of the antisense strand). For example, two residues of thesense strand (located within the mismatch-tolerant region of the sensestrand) that form mismatched base pairs with the corresponding antisensestrand sequence can occur with zero, one, two, three, four or fivematched base pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 4 and 8, but not at positions 5,6 and 7, the mismatched residues of sense strand positions 3 and 4 areadjacent to one another, while the mismatched residues of sense strandpositions 4 and 8 are interspersed by three nucleotides that formmatched base pairs with corresponding residues of the antisense strand).For example, three residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two, three or four matched base pairs located between any twoof these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of thesense strand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 5, 7 and 8, but not at positions4 and 6, the mismatched residues of sense strand positions 7 and 8 areadjacent to one another, while the mismatched residues of sense strandpositions 3 and 5 are interspersed by one nucleotide that forms amatched base pair with the corresponding residue of the antisensestrand—similarly, the mismatched residues of sense strand positions 5and 7 are also interspersed by one nucleotide that forms a matched basepair with the corresponding residue of the antisense strand). Forexample, four residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two or three matched base pairs located between any two ofthese mismatched base pairs.

In another embodiment, for example with numbering also as depicted inFIG. 1, a DsiRNAmm of the invention comprises a mismatch tolerant regionwhich possesses a single mismatched base pair nucleotide at any one ofpositions 17, 18, 19, 20, 21, 22 or 23 of the antisense strand of theDsiRNA (where position 1 is the 5′ terminal nucleotide of the antisensestrand and position 17 is the nucleotide residue of the antisense strandthat is immediately 3′ (downstream) in the antisense strand of theprojected Agog cut site of the target KRAS RNA sequence sufficientlycomplementary to the antisense strand sequence). In certain embodiments,for a DsiRNAmm that possesses a mismatched base pair nucleotide at anyof positions 17, 18, 19, 20, 21, 22 or 23 of the antisense strand withrespect to the sense strand of the DsiRNAmm, the mismatched base pairnucleotide of the antisense strand not only forms a mismatched base pairwith the DsiRNAmm sense strand sequence, but also forms a mismatchedbase pair with a DsiRNAmm target KRAS RNA sequence (thus,complementarity between the antisense strand sequence and the sensestrand sequence is disrupted at the mismatched base pair within theDsiRNAmm, and complementarity is similarly disrupted between theantisense strand sequence of the DsiRNAmm and the target KRAS RNAsequence). In alternative embodiments, the mismatch base pair nucleotideof the antisense strand of a DsiRNAmm only forms a mismatched base pairwith a corresponding nucleotide of the sense strand sequence of theDsiRNAmm, yet base pairs with its corresponding target KRAS RNA sequencenucleotide (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, yet complementarity is maintained between theantisense strand sequence of the DsiRNAmm and the target KRAS RNAsequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 17, 18, 19, 20,21, 22 or 23 of the antisense strand) can further include one, two oreven three additional mismatched base pairs. In preferred embodiments,these one, two or three additional mismatched base pairs of the DsiRNAmmoccur at position(s) 17, 18, 19, 20, 21, 22 and/or 23 of the antisensestrand (and at corresponding residues of the sense strand). In oneembodiment where one additional mismatched base pair is present within aDsiRNAmm, the two mismatched base pairs of the antisense strand canoccur, e.g., at nucleotides of both position 18 and position 20 of theantisense strand (with mismatch also occurring at correspondingnucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 17 and 20, but not at positions 18 and 19, the mismatchedresidues of antisense strand positions 17 and 20 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 17, 18 and 22, but not at positions19, 20 and 21, the mismatched residues of antisense strand positions 17and 18 are adjacent to one another, while the mismatched residues ofantisense strand positions 18 and 122 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 18, 20, 22 and 23, but not atpositions 19 and 21, the mismatched residues of antisense strandpositions 22 and 23 are adjacent to one another, while the mismatchedresidues of antisense strand positions 18 and 20 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the mismatched residues of antisensestrand positions 20 and 22 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

For reasons of clarity, the location(s) of mismatched nucleotideresidues within the above DsiRNAmm agents are numbered in reference tothe 5′ terminal residue of either sense or antisense strands of theDsiRNAmm. The numbering of positions located within themismatch-tolerant region (mismatch region) of the antisense strand canshift with variations in the proximity of the 5′ terminus of the senseor antisense strand to the projected Ago2 cleavage site. Thus, thelocation(s) of preferred mismatch sites within either antisense strandor sense strand can also be identified as the permissible proximity ofsuch mismatches to the projected Ago2 cut site. Accordingly, in onepreferred embodiment, the position of a mismatch nucleotide of the sensestrand of a DsiRNAmm is the nucleotide residue of the sense strand thatis located immediately 5′ (upstream) of the projected Ago2 cleavage siteof the corresponding target KRAS RNA sequence. In other preferredembodiments, a mismatch nucleotide of the sense strand of a DsiRNAmm ispositioned at the nucleotide residue of the sense strand that is locatedtwo nucleotides 5′ (upstream) of the projected Ago2 cleavage site, threenucleotides 5′ (upstream) of the projected Ago2 cleavage site, fournucleotides 5′ (upstream) of the projected Ago2 cleavage site, fivenucleotides 5′ (upstream) of the projected Ago2 cleavage site, sixnucleotides 5′ (upstream) of the projected Ago2 cleavage site, sevennucleotides 5′ (upstream) of the projected Ago2 cleavage site, eightnucleotides 5′ (upstream) of the projected Ago2 cleavage site, or ninenucleotides 5′ (upstream) of the projected Ago2 cleavage site.

Exemplary single mismatch-containing 25/27mer DsiRNAs (DsiRNAmm) includethe following structures (such mismatch-containing structures may alsobe incorporated into other exemplary DsiRNA structures shown herein).

5′-pXX^(M)XXXXXXXXXXXXXXXXXXXXDD-3′3′-XXXX_(M)XXXXXXXXXXXXXXXXXXXXXXp-5′5′-pXXX^(M)XXXXXXXXXXXXXXXXXXXDD-3′3′-XXXXX_(M)XXXXXXXXXXXXXXXXXXXXXp-5′5′-pXXXX^(M)XXXXXXXXXXXXXXXXXXDD-3′3′-XXXXXX_(M)XXXXXXXXXXXXXXXXXXXXp-5′5′-pXXXXX^(M)XXXXXXXXXXXXXXXXXDD-3′3′-XXXXXXX_(M)XXXXXXXXXXXXXXXXXXXp-5′5′-pXXXXXX^(M)XXXXXXXXXXXXXXXXDD-3′3′-XXXXXXXX_(M)XXXXXXXXXXXXXXXXXXp-5′5′-pXXXXXXX^(M)XXXXXXXXXXXXXXXDD-3′3′-XXXXXXXXX_(M)XXXXXXXXXXXXXXXXXp-5′5′-pXXXXXXXX^(M)XXXXXXXXXXXXXXDD-3′3′-XXXXXXXXXX_(M)XXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “D”=DNA, “p”=a phosphate group, “M”=Nucleic acidresidues (RNA, DNA or non-natural or modified nucleic acids) that do notbase pair (hydrogen bond) with corresponding “M” residues of otherwisecomplementary strand when strands are annealed. Any of the residues ofsuch agents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown above, canalso be used in the above DsiRNAmm agents. For the above mismatchstructures, the top strand is the sense strand, and the bottom strand isthe antisense strand.

In certain embodiments, a DsiRNA of the invention can contain mismatchesthat exist in reference to the target KRAS RNA sequence yet do notnecessarily exist as mismatched base pairs within the two strands of theDsiRNA—thus, a DsiRNA can possess perfect complementarity between firstand second strands of a DsiRNA, yet still possess mismatched residues inreference to a target KRAS RNA (which, in certain embodiments, may beadvantageous in promoting efficacy and/or potency and/or duration ofeffect). In certain embodiments, where mismatches occur betweenantisense strand and target KRAS RNA sequence, the position of amismatch is located within the antisense strand at a position(s) thatcorresponds to a sequence of the sense strand located 5′ of theprojected Ago2 cut site of the target region—e.g., antisense strandresidue(s) positioned within the antisense strand to the 3′ of theantisense residue which is complementary to the projected Ago2 cut siteof the target sequence.

Exemplary 25/27mer DsiRNAs that harbor a single mismatched residue inreference to target sequences include the following preferredstructures.

Target RNA Sequence: 5′-. . . XXAXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXBXXXXXXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXEXXXXXXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXAXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXBXXXXXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXEXXXXXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXXAXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXXBXXXXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXEXXXXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXXXAXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXXXBXXXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXEXXXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXXXXAXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXXXXBXXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXEXXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXXXXXAXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXXXXXBXXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXXEXXXXXXXXXXXXXXXXXp-5′Target RNA Sequence: 5′-. . . XXXXXXXXAXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-pXXXXXXXXBXXXXXXXXXXXXXXDD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXXXEXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “D”=DNA, “p”=a phosphate group, “E”=Nucleic acidresidues (RNA, DNA or non-natural or modified nucleic acids) that do notbase pair (hydrogen bond) with corresponding “A” RNA residues ofotherwise complementary (target) strand when strands are annealed, yetoptionally do base pair with corresponding “B” residues (“B” residuesare also RNA, DNA or non-natural or modified nucleic acids). Any of theresidues of such agents can optionally be 2′-O-methyl RNAmonomers—alternating positioning of 2′-O-methyl RNA monomers thatcommences from the 3′-terminal residue of the bottom (second) strand, asshown above, can also be used in the above DsiRNA agents.

In addition to the above-exemplified structures, DsiRNAs of theinvention can also possess one, two or three additional residues thatform further mismatches with the target KRAS RNA sequence. Suchmismatches can be consecutive, or can be interspersed by nucleotidesthat form matched base pairs with the target KRAS RNA sequence. Whereinterspersed by nucleotides that form matched base pairs, mismatchedresidues can be spaced apart from each other within a single strand atan interval of one, two, three, four, five, six, seven or even eightbase paired nucleotides between such mismatch-forming residues.

As for the above-described DsiRNAmm agents, a preferred location withinDsiRNAs for antisense strand nucleotides that form mismatched base pairswith target KRAS RNA sequence (yet may or may not form mismatches withcorresponding sense strand nucleotides) is within the antisense strandregion that is located 3′ (downstream) of the antisense strand sequencewhich is complementary to the projected Ago2 cut site of the DsiRNA(e.g., in FIG. 1, the region of the antisense strand which is 3′ of theprojected Ago2 cut site is preferred for mismatch-forming residues andhappens to be located at positions 17-23 of the antisense strand for the25/27mer agent shown in FIG. 1). Thus, in one preferred embodiment, theposition of a mismatch nucleotide (in relation to the target KRAS RNAsequence) of the antisense strand of a DsiRNAmm is the nucleotideresidue of the antisense strand that is located immediately 3′(downstream) within the antisense strand sequence of the projected Ago2cleavage site of the corresponding target KRAS RNA sequence. In otherpreferred embodiments, a mismatch nucleotide of the antisense strand ofa DsiRNAmm (in relation to the target KRAS RNA sequence) is positionedat the nucleotide residue of the antisense strand that is located twonucleotides 3′ (downstream) of the corresponding projected Ago2 cleavagesite, three nucleotides 3′ (downstream) of the corresponding projectedAgo2 cleavage site, four nucleotides 3′ (downstream) of thecorresponding projected Ago2 cleavage site, five nucleotides 3′(downstream) of the corresponding projected Ago2 cleavage site, sixnucleotides 3′ (downstream) of the projected Ago2 cleavage site, sevennucleotides 3′ (downstream) of the projected Ago2 cleavage site, eightnucleotides 3′ (downstream) of the projected Ago2 cleavage site, or ninenucleotides 3′ (downstream) of the projected Ago2 cleavage site.

In DsiRNA agents possessing two mismatch-forming nucleotides of theantisense strand (where mismatch-forming nucleotides are mismatchforming in relation to target KRAS RNA sequence), mismatches can occurconsecutively (e.g., at consecutive positions along the antisense strandnucleotide sequence). Alternatively, nucleotides of the antisense strandthat form mismatched base pairs with the target KRAS RNA sequence can beinterspersed by nucleotides that base pair with the target KRAS RNAsequence (e.g., for a DsiRNA possessing mismatch-forming nucleotides atpositions 17 and 20 (starting from the 5′ terminus (position 1) of theantisense strand of the 25/27mer agent shown in FIG. 1), but not atpositions 18 and 19, the mismatched residues of sense strand positions17 and 20 are interspersed by two nucleotides that form matched basepairs with corresponding residues of the target KRAS RNA sequence). Forexample, two residues of the antisense strand (located within themismatch-tolerant region of the antisense strand) that form mismatchedbase pairs with the corresponding target KRAS RNA sequence can occurwith zero, one, two, three, four or five matched base pairs (withrespect to target KRAS RNA sequence) located between thesemismatch-forming base pairs.

For certain DsiRNAs possessing three mismatch-forming base pairs(mismatch-forming with respect to target KRAS RNA sequence),mismatch-forming nucleotides can occur consecutively (e.g., in a tripletalong the antisense strand nucleotide sequence). Alternatively,nucleotides of the antisense strand that form mismatched base pairs withthe target KRAS RNA sequence can be interspersed by nucleotides thatform matched base pairs with the target KRAS RNA sequence (e.g., for aDsiRNA possessing mismatched nucleotides at positions 17, 18 and 22, butnot at positions 19, 20 and 21, the mismatch-forming residues ofantisense strand positions 17 and 18 are adjacent to one another, whilethe mismatch-forming residues of antisense strand positions 18 and 22are interspersed by three nucleotides that form matched base pairs withcorresponding residues of the target KRAS RNA). For example, threeresidues of the antisense strand (located within the mismatch-tolerantregion of the antisense strand) that form mismatched base pairs with thecorresponding target KRAS RNA sequence can occur with zero, one, two,three or four matched base pairs located between any two of thesemismatch-forming base pairs.

For certain DsiRNAs possessing four mismatch-forming base pairs(mismatch-forming with respect to target KRAS RNA sequence),mismatch-forming nucleotides can occur consecutively (e.g., in aquadruplet along the sense strand nucleotide sequence). Alternatively,nucleotides of the antisense strand that form mismatched base pairs withthe target KRAS RNA sequence can be interspersed by nucleotides thatform matched base pairs with the target KRAS RNA sequence (e.g., for aDsiRNA possessing mismatch-forming nucleotides at positions 17, 19, 21and 22, but not at positions 18 and 20, the mismatch-forming residues ofantisense strand positions 21 and 22 are adjacent to one another, whilethe mismatch-forming residues of antisense strand positions 17 and 19are interspersed by one nucleotide that forms a matched base pair withthe corresponding residue of the target KRAS RNA sequence—similarly, themismatch-forming residues of antisense strand positions 19 and 21 arealso interspersed by one nucleotide that forms a matched base pair withthe corresponding residue of the target KRAS RNA sequence). For example,four residues of the antisense strand (located within themismatch-tolerant region of the antisense strand) that form mismatchedbase pairs with the corresponding target KRAS RNA sequence can occurwith zero, one, two or three matched base pairs located between any twoof these mismatch-forming base pairs.

The above DsiRNAmm and other DsiRNA structures are described in order toexemplify certain structures of DsiRNAmm and DsiRNA agents. Design ofthe above DsiRNAmm and DsiRNA structures can be adapted to generate,e.g., DsiRNAmm forms of other DsiRNA structures shown infra. Asexemplified above, DsiRNAs can also be designed that possess singlemismatches (or two, three or four mismatches) between the antisensestrand of the DsiRNA and a target sequence, yet optionally can retainperfect complementarity between sense and antisense strand sequences ofa DsiRNA.

It is further noted that the DsiRNA agents exemplified infra can alsopossess insertion/deletion (in/del) structures within theirdouble-stranded and/or target KRAS RNA-aligned structures. Accordingly,the DsiRNAs of the invention can be designed to possess in/delvariations in, e.g., antisense strand sequence as compared to targetKRAS RNA sequence and/or antisense strand sequence as compared to sensestrand sequence, with preferred location(s) for placement of such in/delnucleotides corresponding to those locations described above forpositioning of mismatched and/or mismatch-forming base pairs.

It is also noted that the DsiRNAs of the instant invention can toleratemismatches within the 3′-terminal region of the sense strand/5′-terminalregion of the antisense strand, as this region is modeled to beprocessed by Dicer and liberated from the guide strand sequence thatloads into RISC. Exemplary DsiRNA structures of the invention thatharbor such mismatches include the following:

Target RNA Sequence: 5′-. . . XXXXXXXXXXXXXXXXXXXXXHXXX . . .-3′DsiRNA Sense Strand: 5′-pXXXXXXXXXXXXXXXXXXXXXIXDD-3′DsiRNA Antisense Strand: 3′ -XXXXXXXXXXXXXXXXXXXXXXXJXXXp-5′Target RNA Sequence: 5′-. . . XXXXXXXXXXXXXXXXXXXXXXHXX. . .-3′DsiRNA Sense Strand: 5′-pXXXXXXXXXXXXXXXXXXXXXXIDD-3′DsiRNA Antisense Strand: 3′-XXXXXXXXXXXXXXXXXXXXXXXXJXXp-5′Target RNA Sequence: 5′-. . . XXXXXXXXXXXXXXXXXXXXXXXHX . . .-3′DsiRNA Sense Strand: 5′-pXXXXXXXXXXXXXXXXXXXXXXXID-3′DsiRNA Antisense Strand: 3′-XXXXXXXXXXXXXXXXXXXXXXXXXJXp-5′Target RNA Sequence: 5′-. . . XXXXXXXXXXXXXXXXXXXXXXXXH . . .-3′DsiRNA Sense Strand: 5′-pXXXXXXXXXXXXXXXXXXXXXXXDI-3′DsiRNA Antisense Strand: 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXJp-5′wherein “X”=RNA, “D”=DNA, “p”=a phosphate group, “I” and “J”=Nucleicacid residues (RNA, DNA or non-natural or modified nucleic acids) thatdo not base pair (hydrogen bond) with one another, yet optionally “J” iscomplementary to target RNA sequence nucleotide “H”. Any of the residuesof such agents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown above—or anyof the above-described methylation patterns—can also be used in theabove DsiRNA agents. The above mismatches can also be combined withinthe DsiRNAs of the instant invention.

In the below structures, such mismatches are introduced within theasymmetric KRAS-420 DsiRNA (newly-introduced mismatch residues areitalicized):

KRAS-420 25/27mer DsiRNA, mismatch position=22 of sense strand (from5′-terminus)

  5′-GUAUUUGCCAUAAAUAAUACU^(C)Aat-3′ (SEQ ID NO: 5136)3′-CACAUAAACGGUAUUUAUUAUGA_(U)UUA-5′ (SEQ ID NO: 26)Optionally, the mismatched “C” residue of position 22 of the sensestrand is alternatively “G” or “U”,KRAS-420 25/27mer DsiRNA, mismatch position=23 of sense strand

  5′-GUAUUUGCCAUAAAUAAUACUA^(C)at-3′ (SEQ ID NO: 5137)3′-CACAUAAACGGUAUUUAUUAUGAU_(U)UA-5′ (SEQ ID NO: 26)

Optionally, the mismatched “C” residue of position 23 of the sensestrand is alternatively “G” or “U”.

KRAS-420 25/27mer DsiRNA, mismatch position=24 of sense strand

  5′-GUAUUUGCCAUAAAUAAUACUAA^(c)t-3′ (SEQ ID NO: 5138)3′-CACAUAAACGGUAUUUAUUAUGAUU_(U)A-5′ (SEQ ID NO: 26)Optionally, the mismatched “c” residue of position 24 of the sensestrand is alternatively “g” or “t”.KRAS-420 25/27mer DsiRNA, mismatch position=25 of sense strand

  5′-GUAUUUGCCAUAAAUAAUACUAAa^(g)-3′ (SEQ ID NO: 5139)3′-CACAUAAACGGUAUUUAUUAUGAUUU_(A)-5′ (SEQ ID NO: 26)Optionally, the mismatched “g” residue of position 25 of the sensestrand is alternatively “c” or “a”.KRAS-420 25/27mer DsiRNA, mismatch position=1 of antisense strand

  5′-GUAUUUGCCAUAAAUAAUACUAAa^(t)-3′ (SEQ ID NO: 105)3′-CACAUAAACGGUAUUUAUUAUGAUUU_(C)-5′ (SEQ ID NO: 5140)Optionally, the mismatched “C” residue of position 1 of the antisensestrand is alternatively “G” or “U”.KRAS-420 25/27mer DsiRNA, mismatch position=2 of antisense strand

  5′-GUAUUUGCCAUAAAUAAUACUAA^(a)t-3′ (SEQ ID NO: 105)3′-CACAUAAACGGUAUUUAUUAUGAUU_(C)A-5′ (SEQ ID NO: 5141)Optionally, the mismatched “C” residue of position 2 of the antisensestrand is alternatively “G” or “A”.KRAS-420 25/27mer DsiRNA, mismatch position=3 of antisense strand

  5′-GUAUUUGCCAUAAAUAAUACUA^(A)at-3′ (SEQ ID NO: 105)3′-CACAUAAACGGUAUUUAUUAUGAU_(G)UA-5′. (SEQ ID NO: 5142)Optionally, the mismatched “G” residue of position 3 of the antisensestrand is alternatively “A” or “C”KRAS-420 25/27mer DsiRNA, mismatch position=4 of antisense strand

  5′-GUAUUUGCCAUAAAUAAUACU^(A)Aat-3′ (SEQ ID NO: 105)3′-CACAUAAACGGUAUUUAUUAUGA_(G)UUA-5′ (SEQ ID NO: 5143)Optionally, the mismatched “G” residue of position 4 of the antisensestrand is alternatively “A” or “C”.

As noted above, introduction of such mismatches can be performed uponany of the DsiRNAs described herein.

The mismatches of such DsiRNA structures can be combined to produce aDsiRNA possessing, e.g., two, three or even four mismatches within the3′-terminal four nucleotides of the sense strand/5′-terminal fournucleotides of the antisense strand.

Indeed, in view of the flexibility of sequences which can beincorporated into DsiRNAs at the 3′-terminal residues of the sensestrand/5′-terminal residues of the antisense strand, in certainembodiments, the sequence requirements of an asymmetric DsiRNA of theinstant invention can be represented as the following (minimalist)structure (shown for an exemplary KRAS-420 DsiRNA sequence):

  5′-GUAUUUGCCAUAAAUAAUACUXXX[X]_(n)-3′ (SEQ ID NO: 6839)3′-CACAUAAACGGUAUUUAUUAUXXXXX[X]_(n)-5′ (SEQ ID NO: 6840)where n=1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 50, or 1 to 80 or more.KRAS-420 Target:

(SEQ ID NO: 6841) 5′-GTGTATTTGCCATAAATAATAXXXXXX-3′

The KRAS target sight may also be a site which is targeted by one ormore of several oligonucleotides whose complementary target sitesoverlap with a stated target site. For example, for the exemplaryKRAS-420 DsiRNA, it is noted that certain DsiRNAs targeting overlappingand only slightly offset KRAS sequences can exhibit activity levelssimilar to that of KRAS-420 (specifically, see KRAS-415, KRAS-417 andKRAS-418 DsiRNAs of FIG. 10, and KRAS-422, KRAS-423, KRAS-424, KRAS-425and KRAS-426 DsiRNAs of Table 9 and FIGS. 12 and 13. Thus, in certainembodiments, a designated target sequence region can be effectivelytargeted by a series of DsiRNAs possessing largely overlappingsequences. (E.g., if considering DsiRNAs surrounding the KRAS-420 site,a more encompassing KRAS target sequence might be recited as, e.g.,5′-TCTTTGTGTATTTGCCATAAATAATACTAAATCA-3′ (SEQ ID NO: 6842), wherein anygiven DsiRNA (e.g., a DsiRNA selected from KRAS-415, KRAS-417, KRAS-418,KRAS-420, KRAS-422, KRAS-423, KRAS-424, KRAS-425 and KRAS-426) onlytargets a sub-sequence within such a sequence region, yet the entiresequence can be considered a viable target for such a series ofDsiRNAs).

Additionally and/or alternatively, mismatches within the 3′-terminalfour nucleotides of the sense strand/5′-terminal four nucleotides of theantisense strand can be combined with mismatches positioned at othermismatch-tolerant positions, as described above.

In view of the present identification of the above-described Dicersubstrate agents (DsiRNAs) as inhibitors of KRAS levels via targeting ofspecific KRAS sequences, it is also recognized that DsiRNAs havingstructures similar to those described herein can also be synthesizedwhich target other sequences within the KRAS sequence of SEQ ID NO: 1 orSEQ ID NO: 3, or within variants thereof (e.g., target sequencespossessing 80% identity, 90% identity, 95% identity, 96% identity, 97%identity, 98% identity, 99% or more identity to a sequence of SEQ ID NO:1 and/or SEQ ID NO: 3).

Anti-KRAS DsiRNA Design/Synthesis

It has been found empirically that longer dsRNA species of from 25 to 35nucleotides (DsiRNAs) and especially from 25 to 30 nucleotides giveunexpectedly effective results in terms of potency and duration ofaction, as compared to 19-23mer siRNA agents. Without wishing to bebound by the underlying theory of the dsRNA processing mechanism, it isthought that the longer dsRNA species serve as a substrate for the Dicerenzyme in the cytoplasm of a cell. In addition to cleaving the dsRNA ofthe invention into shorter segments, Dicer is thought to facilitate theincorporation of a single-stranded cleavage product derived from thecleaved dsRNA into the RISC complex that is responsible for thedestruction of the cytoplasmic RNA (e.g., KRAS RNA) of or derived fromthe target gene, KRAS (or other gene associated with a KRAS-associateddisease or disorder). Prior studies (Rossi et al., U.S. PatentApplication No. 2007/0265220) have shown that the cleavability of adsRNA species (specifically, a DsiRNA agent) by Dicer corresponds withincreased potency and duration of action of the dsRNA species.

Certain preferred anti-KRAS DsiRNA agents were selected from apre-screened population. Design of DsiRNAs can optionally involve use ofpredictive scoring algorithms that perform in silico assessments of theprojected activity/efficacy of a number of possible DsiRNA agentsspanning a region of sequence. Information regarding the design of suchscoring algorithms can be found, e.g., in Gong et al. (BMCBioinformatics 2006, 7:516), though a more recent “v3” algorithmrepresents a theoretically improved algorithm relative to siRNA scoringalgorithms previously available in the art. (The “v3” scoring algorithmis a machine learning algorithm that is not reliant upon any biases inhuman sequence. In addition, the “v3” algorithm derives from a data setthat is approximately three-fold larger than that from which an older“v2” algorithm such as that described in Gong et al. derives.)

The first and second oligonucleotides of the DsiRNA agents of theinstant invention are not required to be completely complementary. Infact, in one embodiment, the 3′-terminus of the sense strand containsone or more mismatches. In one aspect, two mismatches are incorporatedat the 3′ terminus of the sense strand. In another embodiment, theDsiRNA of the invention is a double stranded RNA molecule containing twoRNA oligonucleotides each of which is 27 nucleotides in length and, whenannealed to each other, have blunt ends and a two nucleotide mismatch onthe 3′-terminus of the sense strand (the 5′-terminus of the antisensestrand). The use of mismatches or decreased thermodynamic stability(specifically at the 3′-sense/5′-antisense position) has been proposedto facilitate or favor entry of the antisense strand into RISC (Schwarzet al., 2003, Cell 115: 199-208; Khvorova et al., 2003, Cell 115:209-216), presumably by affecting some rate-limiting unwinding stepsthat occur with entry of the siRNA into RISC. Thus, terminal basecomposition has been included in design algorithms for selecting active21mer siRNA duplexes (Ui-Tei et al., 2004, Nucleic Acids Res 32:936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330). With Dicercleavage of the dsRNA of this embodiment, the small end-terminalsequence which contains the mismatches will either be left unpaired withthe antisense strand (become part of a 3′-overhang) or be cleavedentirely off the final 21-mer siRNA. These “mismatches”, therefore, donot persist as mismatches in the final RNA component of RISC. Thefinding that base mismatches or destabilization of segments at the3′-end of the sense strand of Dicer substrate improved the potency ofsynthetic duplexes in RNAi, presumably by facilitating processing byDicer, was a surprising finding of past works describing the design anduse of 25-30mer dsRNAs (also termed “DsiRNAs” herein; Rossi et al., U.S.Patent Application Nos. 2005/0277610, 2005/0244858 and 2007/0265220).

Modification of Anti-KRAS DsiRNAs

One major factor that inhibits the effect of double stranded RNAs(“dsRNAs”) is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) bynucleases. A 3′-exonuclease is the primary nuclease activity present inserum and modification of the 3′-ends of antisense DNA oligonucleotidesis crucial to prevent degradation (Eder et al., 1991, Antisense Res Dev,1: 141-151). An RNase-T family nuclease has been identified called ERI-1which has 3′ to 5′ exonuclease activity that is involved in regulationand degradation of siRNAs (Kennedy et al., 2004, Nature 427: 645-649;Hong et al., 2005, Biochem J, 390: 675-679). This gene is also known asThex1 (NM_(—)02067) in mice or THEX1 (NM_(—)153332) in humans and isinvolved in degradation of histone mRNA; it also mediates degradation of3′-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al.,2006, J Biol Chem, 281: 30447-30454). It is therefore reasonable toexpect that 3′-end-stabilization of dsRNAs, including the DsiRNAs of theinstant invention, will improve stability.

XRN1 (NM_(—)019001) is a 5′ to 3′ exonuclease that resides in P-bodiesand has been implicated in degradation of mRNA targeted by miRNA(Rehwinkel et al., 2005, RNA 11: 1640-1647) and may also be responsiblefor completing degradation initiated by internal cleavage as directed bya siRNA. XRN2 (NM_(—)012255) is a distinct 5′ to 3′ exonuclease that isinvolved in nuclear RNA processing.

RNase A is a major endonuclease activity in mammals that degrades RNAs.It is specific for ssRNA and cleaves at the 3′-end of pyrimidine bases.SiRNA degradation products consistent with RNase A cleavage can bedetected by mass spectrometry after incubation in serum (Turner et al.,2007, Mol Biosyst 3: 43-50). The 3′-overhangs enhance the susceptibilityof siRNAs to RNase degradation. Depletion of RNase A from serum reducesdegradation of siRNAs; this degradation does show some sequencepreference and is worse for sequences having poly A/U sequence on theends (Haupenthal et al., 2006 Biochem Pharmacol 71: 702-710). Thissuggests the possibility that lower stability regions of the duplex may“breathe” and offer transient single-stranded species available fordegradation by RNase A. RNase A inhibitors can be added to serum andimprove siRNA longevity and potency (Haupenthal et al., 2007, Int J.Cancer 121: 206-210).

In 21 mers, phosphorothioate or boranophosphate modifications directlystabilize the internucleoside phosphate linkage. Boranophosphatemodified RNAs are highly nuclease resistant, potent as silencing agents,and are relatively non-toxic. Boranophosphate modified RNAs cannot bemanufactured using standard chemical synthesis methods and instead aremade by in vitro transcription (NT) (Hall et al., 2004, Nucleic AcidsRes 32: 5991-6000; Hall et al., 2006, Nucleic Acids Res 34: 2773-2781).Phosphorothioate (PS) modifications can be easily placed in the RNAduplex at any desired position and can be made using standard chemicalsynthesis methods. The PS modification shows dose-dependent toxicity, somost investigators—have recommended limited incorporation in siRNAs,favoring the 3′-ends where protection from nucleases is most important(Harborth et al., 2003, Antisense Nucleic Acid Drug Dev 13: 83-105; Chiuand Rana, 2003, Mol Cell 10: 549-561; Braasch et al., 2003, Biochemistry42: 7967-7975; Amarzguioui et al., 2003, Nucleic Acids Research 31:589-595). More extensive PS modification can be compatible with potentRNAi activity; however, use of sugar modifications (such as 2′-O-methylRNA) may be superior (Choung et al., 2006, Biochem Biophys Res Commun342: 919-927).

A variety of substitutions can be placed at the 2′-position of theribose which generally increases duplex stability (T_(m)) and cangreatly improve nuclease resistance. 2′-O-methyl RNA is a naturallyoccurring modification found in mammalian ribosomal RNAs and transferRNAs. 2′-O-methyl modification in siRNAs is known, but the preciseposition of modified bases within the duplex is important to retainpotency and complete substitution of 2′-O-methyl RNA for RNA willinactivate the siRNA. For example, a pattern that employs alternating2′-O-methyl bases can have potency equivalent to unmodified RNA and isquite stable in serum (Choung et al., 2006, Biochem Biophys Res Commun342: 919-927; Czauderna et al., 2003, Nucleic Acids Research 31:2705-2716).

The 2′-fluoro (2′-F) modification is also compatible with dsRNA (e.g.,siRNA and DsiRNA) function; it is most commonly placed at pyrimidinesites (due to reagent cost and availability) and can be combined with2′-O-methyl modification at purine positions; 2′-F purines are availableand can also be used. Heavily modified duplexes of this kind can bepotent triggers of RNAi in vitro (Allerson et al., 2005, J Med Chem 48:901-904; Prakash et al., 2005, J Med Chem 48: 4247-4253; Kraynack andBaker, 2006, RNA 12: 163-176) and can improve performance and extendduration of action when used in vivo (Morrissey et al., 2005, Hepatology41: 1349-1356; Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007). Ahighly potent, nuclease stable, blunt 19mer duplex containingalternative 2′-F and 2′-O-Me bases is taught by Allerson. In thisdesign, alternating 2′-O-Me residues are positioned in an identicalpattern to that employed by Czauderna, however the remaining RNAresidues are converted to 2′-F modified bases. A highly potent, nucleaseresistant siRNA employed by Morrissey employed a highly potent, nucleaseresistant siRNA in vivo. In addition to 2′-O-Me RNA and 2′-F RNA, thisduplex includes DNA, RNA, inverted abasic residues, and a 3′-terminal PSinternucleoside linkage. While extensive modification has certainbenefits, more limited modification of the duplex can also improve invivo performance and is both simpler and less costly to manufacture.Soutschek et al. (2004, Nature 432: 173-178) employed a duplex in vivoand was mostly RNA with two 2′-O-Me RNA bases and limited 3′-terminal PSinternucleoside linkages.

Locked nucleic acids (LNAs) are a different class of 2′-modificationthat can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patternsof LNA incorporation that retain potency are more restricted than2′-O-methyl or 2′-F bases, so limited modification is preferred (Braaschet al., 2003, Biochemistry 42: 7967-7975; Grunweller et al., 2003,Nucleic Acids Res 31: 3185-3193; Elmen et al., 2005, Nucleic Acids Res33: 439-447). Even with limited incorporation, the use of LNAmodifications can improve dsRNA performance in vivo and may also alteror improve off target effect profiles (Mook et al., 2007, Mol CancerTher 6: 833-843).

Synthetic nucleic acids introduced into cells or live animals can berecognized as “foreign” and trigger an immune response. Immunestimulation constitutes a major class of off-target effects which candramatically change experimental results and even lead to cell death.The innate immune system includes a collection of receptor moleculesthat specifically interact with DNA and RNA that mediate theseresponses, some of which are located in the cytoplasm and some of whichreside in endosomes (Marques and Williams, 2005, Nat Biotechnol 23:1399-1405; Schlee et al., 2006, Mol Ther 14: 463-470). Delivery ofsiRNAs by cationic lipids or liposomes exposes the siRNA to bothcytoplasmic and endosomal compartments, maximizing the risk fortriggering a type 1 interferon (IFN) response both in vitro and in vivo(Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007; Sioud andSorensen, 2003, Biochem Biophys Res Commun 312: 1220-1225; Sioud, 2005,J Mol Biol 348: 1079-1090; Ma et al., 2005, Biochem Biophys Res Commun330: 755-759). RNAs transcribed within the cell are less immunogenic(Robbins et al., 2006, Nat Biotechnol 24: 566-571) and synthetic RNAsthat are immunogenic when delivered using lipid-based methods can evadeimmune stimulation when introduced unto cells by mechanical means, evenin vivo (Heidel et al., 2004, Nat Biotechnol 22: 1579-1582). However,lipid based delivery methods are convenient, effective, and widely used.Some general strategy to prevent immune responses is needed, especiallyfor in vivo application where all cell types are present and the risk ofgenerating an immune response is highest. Use of chemically modifiedRNAs may solve most or even all of these problems.

In certain embodiments, modifications can be included in the anti-KRASDsiRNA agents of the present invention so long as the modification doesnot prevent the DsiRNA agent from possessing KRAS inhibitory activity.In one embodiment, one or more modifications are made that enhance Dicerprocessing of the DsiRNA agent (an assay for determining Dicerprocessing of a DsiRNA is described supra). In a second embodiment, oneor more modifications are made that result in more effective KRASinhibition (as described herein, KRAS inhibition/KRAS inhibitoryactivity of a DsiRNA can be assayed via art-recognized methods fordetermining RNA levels, or for determining Kras polypeptide levels,should such levels be assessed in lieu of or in addition to assessmentof, e.g., KRAS mRNA levels). In a third embodiment, one or moremodifications are made that support greater KRAS inhibitory activity(means of determining KRAS inhibitory activity are described supra). Ina fourth embodiment, one or more modifications are made that result ingreater potency of KRAS inhibitory activity per each DsiRNA agentmolecule to be delivered to the cell (potency of KRAS inhibitoryactivity is described supra). Modifications can be incorporated in the3′-terminal region, the 5′-terminal region, in both the 3′-terminal and5′-terminal region or in some instances in various positions within thesequence. With the restrictions noted above in mind, numbers andcombinations of modifications can be incorporated into the DsiRNA agent.Where multiple modifications are present, they may be the same ordifferent. Modifications to bases, sugar moieties, the phosphatebackbone, and their combinations are contemplated. Either 5′-terminuscan be phosphorylated.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003,Nucleic Acids Research 31: 589-595). Examples of modificationscontemplated for the base groups include abasic sugars, 2-O-alkylmodified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's,could also be incorporated. Many other modifications are known and canbe used so long as the above criteria are satisfied. Examples ofmodifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988and 6,291,438 and in U.S. published patent application No. 2004/0203145A1. Other modifications are disclosed in Herdewijn (2000, AntisenseNucleic Acid Drug Dev 10: 297-310), Eckstein (2000, Antisense NucleicAcid Drug Dev 10: 117-21), Rusckowski et al. (2000, Antisense NucleicAcid Drug Dev 10: 333-345), Stein et al. (2001, Antisense Nucleic AcidDrug Dev 11: 317-25); Vorobjev et al. (2001, Antisense Nucleic Acid DrugDev 11: 77-85).

One or more modifications contemplated can be incorporated into eitherstrand. The placement of the modifications in the DsiRNA agent cangreatly affect the characteristics of the DsiRNA agent, includingconferring greater potency and stability, reducing toxicity, enhanceDicer processing, and minimizing an immune response. In one embodiment,the antisense strand or the sense strand or both strands have one ormore 2′-O-methyl modified nucleotides. In another embodiment, theantisense strand contains 2′-O-methyl modified nucleotides. In anotherembodiment, the antisense stand contains a 3′ overhang that is comprisedof 2′-O-methyl modified nucleotides. The antisense strand could alsoinclude additional 2′-O-methyl modified nucleotides.

In certain embodiments of the present invention, the anti-KRAS DsiRNAagent has one or more of the following properties: (i) the DsiRNA agentis asymmetric, e.g., has a 3′ overhang on the antisense strand and (ii)the DsiRNA agent has a modified 3′ end on the sense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the longest strand in the dsRNAcomprises 25-35 nucleotides (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 nucleotides). In certain such embodiments, the DsiRNA agent isasymmetric such that the sense strand comprises 25-34 nucleotides andthe 3′ end of the sense strand forms a blunt end with the 5′ end of theantisense strand while the antisense strand comprises 26-35 nucleotidesand forms an overhang on the 3′ end of the antisense strand. In oneembodiment, the DsiRNA agent is asymmetric such that the sense strandcomprises 25-28 nucleotides and the antisense strand comprises 25-30nucleotides. Thus, the resulting dsRNA has an overhang on the 3′ end ofthe antisense strand. The overhang is 1-4 nucleotides, for example 2nucleotides. The sense strand may also have a 5′ phosphate.

In other embodiments, the sense strand of the DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing. Suitable modifiers include nucleotidessuch as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotidesand the like and sterically hindered molecules, such as fluorescentmolecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in dNMPs. Other nucleotides modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the sense strand.When sterically hindered molecules are utilized, they are attached tothe ribonucleotide at the 3′ end of the antisense strand. Thus, thelength of the strand does not change with the incorporation of themodifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the DsiRNA agent to direct the orientationof Dicer processing of the antisense strand. In a further embodiment ofthe present invention, two terminal DNA bases are substituted for tworibonucleotides on the 3′-end of the sense strand forming a blunt end ofthe duplex on the 3′ end of the sense strand and the 5′ end of theantisense strand, and a two-nucleotide RNA overhang is located on the3′-end of the antisense strand. This is an asymmetric composition withDNA on the blunt end and RNA bases on the overhanging end.

The sense and antisense strands of a DsiRNA agent of the instantinvention anneal under biological conditions, such as the conditionsfound in the cytoplasm of a cell. In addition, a region of one of thesequences, particularly of the antisense strand, of the DsiRNA agent hasa sequence length of at least 19 nucleotides, wherein these nucleotidesare in the 21-nucleotide region adjacent to the 3′ end of the antisensestrand and are sufficiently complementary to a nucleotide sequence ofthe RNA produced from the target gene.

The DsiRNA agent can also have one or more of the following additionalproperties: (a) the antisense strand has a right shift from the typical21mer (e.g., the DsiRNA comprises a length of antisense strandnucleotides that extends to the 5′ of a projected Dicer cleavage sitewithin the DsiRNA, with such antisense strand nucleotides base pairedwith corresponding nucleotides of the sense strand extending 3′ of aprojected Dicer cleavage site in the sense strand), (b) the strands maynot be completely complementary, i.e., the strands may contain simplemismatched base pairs (in certain embodiments, the DsiRNAs of theinvention possess 1, 2, 3, 4 or even 5 or more mismatched base pairs,provided that KRAS inhibitory activity of the DsiRNA possessingmismatched base pairs is retained at sufficient levels (e.g., retains atleast 50% KRAS inhibitory activity or more, at least 60% KRAS inhibitoryactivity or more, at least 70% KRAS inhibitory activity or more, atleast 80% KRAS inhibitory activity or more, at least 90% KRAS inhibitoryactivity or more or at least 95% KRAS inhibitory activity or more ascompared to a corresponding DsiRNA not possessing mismatched base pairs.In certain embodiments, mismatched base pairs exist between theantisense and sense strands of a DsiRNA. In some embodiments, mismatchedbase pairs exist (or are predicted to exist) between the antisensestrand and the target RNA. In certain embodiments, the presence of amismatched base pair(s) between an antisense strand residue and acorresponding residue within the target RNA that is located 3′ in thetarget RNA sequence of a projected Ago2 cleavage site retains and mayeven enhance KRAS inhibitory activity of a DsiRNA of the invention) and(c) base modifications such as locked nucleic acid(s) may be included inthe 5′ end of the sense strand. A “typical” 21 mer siRNA is designedusing conventional techniques. In one technique, a variety of sites arecommonly tested in parallel or pools containing several distinct siRNAduplexes specific to the same target with the hope that one of thereagents will be effective (Ji et al., 2003, FEBS Lett 552: 247-252).Other techniques use design rules and algorithms to increase thelikelihood of obtaining active RNAi effector molecules (Schwarz et al.,2003, Cell 115: 199-208; Khvorova et al., 2003, Cell 115: 209-216;Ui-Tei et al., 2004, Nucleic Acids Res 32: 936-948; Reynolds et al.,2004, Nat Biotechnol 22: 326-330; Krol et al., 2004, J Biol Chem 279:42230-42239; Yuan et al., 2004, Nucl Acids Res 32(Webserver issue):W130-134; Boese et al., 2005, Methods Enzymol 392: 73-96). Highthroughput selection of siRNA has also been developed (U.S. publishedpatent application No. 2005/0042641 A1). Potential target sites can alsobe analyzed by secondary structure predictions (Heale et al., 2005,Nucleic Acids Res 33(3): e30). This 21mer is then used to design a rightshift to include 3-9 additional nucleotides on the 5′ end of the 21mer.The sequence of these additional nucleotides is not restricted. In oneembodiment, the added ribonucleotides are based on the sequence of thetarget gene. Even in this embodiment, full complementarity between thetarget sequence and the antisense siRNA is not required.

The first and second oligonucleotides of a DsiRNA agent of the instantinvention are not required to be completely complementary. They onlyneed to be sufficiently complementary to anneal under biologicalconditions and to provide a substrate for Dicer that produces a siRNAsufficiently complementary to the target sequence. Locked nucleic acids,or LNA's, are well known to a skilled artisan (Elmen et al., 2005,Nucleic Acids Res 33: 439-447; Kurreck et al., 2002, Nucleic Acids Res30: 1911-1918; Crinelli et al., 2002, Nucleic Acids Res 30: 2435-2443;Braasch and Corey, 2001, Chem Biol 8: 1-7; Bondensgaard et al., 2000,Chemistry 6: 2687-2695; Wahlestedt et al., 2000, Proc Natl Acad Sci USA97: 5633-5638). In one embodiment, an LNA is incorporated at the 5′terminus of the sense strand. In another embodiment, an LNA isincorporated at the 5′ terminus of the sense strand in duplexes designedto include a 3′ overhang on the antisense strand.

In certain embodiments, the DsiRNA agent of the instant invention has anasymmetric structure, with the sense strand having a 25-base pairlength, and the antisense strand having a 27-base pair length with a 2base 3′-overhang. In other embodiments, this DsiRNA agent having anasymmetric structure further contains 2 deoxynucleotides at the 3′ endof the sense strand in place of two of the ribonucleotides.

Certain DsiRNA agent compositions containing two separateoligonucleotides can be linked by a third structure. The third structurewill not block Dicer activity on the DsiRNA agent and will not interferewith the directed destruction of the RNA transcribed from the targetgene. In one embodiment, the third structure may be a chemical linkinggroup. Many suitable chemical linking groups are known in the art andcan be used. Alternatively, the third structure may be anoligonucleotide that links the two oligonucleotides of the DsiRNA agentin a manner such that a hairpin structure is produced upon annealing ofthe two oligonucleotides making up the dsRNA composition. The hairpinstructure will not block Dicer activity on the DsiRNA agent and will notinterfere with the directed destruction of the KRAS RNA.

In certain embodiments, the anti-KRAS DsiRNA agent of the invention hasseveral properties which enhance its processing by Dicer. According tosuch embodiments; the DsiRNA agent has a length sufficient such that itis processed by Dicer to produce an siRNA and at least one of thefollowing properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3′overhang on the sense strand and (ii) the DsiRNA agent has a modified 3′end on the antisense strand to direct orientation of Dicer binding andprocessing of the dsRNA to an active siRNA. According to theseembodiments, the longest strand in the DsiRNA agent comprises 25-30nucleotides. In one embodiment, the sense strand comprises 25-30nucleotides and the antisense strand comprises 25-28 nucleotides. Thus,the resulting dsRNA has an overhang on the 3′ end of the sense strand.The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisensestrand may also have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing. Suitable modifiers include nucleotidessuch as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotidesand the like and sterically hindered molecules, such as fluorescentmolecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in dNMPs. Other nucleotide modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the sense strand.When sterically hindered molecules are utilized, they are attached tothe ribonucleotide at the 3′ end of the antisense strand. Thus, thelength of the strand does not change with the incorporation of themodifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the sense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of theantisense strand and the 3′ end of the sense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the antisensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

In certain other embodiments, the antisense strand of a DsiRNA agent ismodified for Dicer processing by suitable modifiers located at the 3′end of the antisense strand, i.e., the DsiRNA agent is designed todirect orientation of Dicer binding and processing. Suitable modifiersinclude nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of thesense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is also an asymmetric composition with DNA on the blunt endand RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the target KRASRNA.

Additionally, the DsiRNA agent structure can be optimized to ensure thatthe oligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention, a 27-bpoligonucleotide of the DsiRNA agent structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand.

US 2007/0265220 discloses that 27mer DsiRNAs show improved stability inserum over comparable 21mer siRNA compositions, even absent chemicalmodification. Modifications of DsiRNA agents, such as inclusion of2′-O-methyl RNA in the antisense strand, in patterns such as detailedabove, when coupled with addition of a 5′ Phosphate, can improvestability of DsiRNA agents. Addition of 5′-phosphate to all strands insynthetic RNA duplexes may be an inexpensive and physiological method toconfer some limited degree of nuclease stability. The chemicalmodification patterns of the DsiRNA agents of the instant invention aredesigned to enhance the efficacy of such agents. Accordingly, suchmodifications are designed to avoid reducing potency of DsiRNA agents;to avoid interfering with Dicer processing of DsiRNA agents; to improvestability in biological fluids (reduce nuclease sensitivity) of DsiRNAagents; or to block or evade detection by the innate immune system. Suchmodifications are also designed to avoid being toxic and to avoidincreasing the cost or impact the ease of manufacturing the instantDsiRNA agents of the invention.

KRAS Biology and Biochemistry

Transformation is a cumulative process whereby normal control of cellgrowth and differentiation is interrupted, usually through theaccumulation of mutations affecting the expression of genes thatregulate cell growth and differentiation.

The platelet derived growth factor (PDGF) system has served as aprototype for identification of substrates of the receptor tyrosinekinases. Certain enzymes become activated by the PDGF receptor kinase,including phospholipase C and phosphatidylinositol 3′ kinase, Rasguanosine triphosphate (GTPase) activating protein (GAP) and src-liketyrosine kinases. GAP regulates the function of the Ras protein bystimulating the GTPase activity of the 21 kD Ras protein. Barbacid, 56Ann. Rev. Biochem. 779, 1987. Microinjection of oncogenically activatedRas into NIH 3T3 cells has been shown to induce DNA synthesis. Mutationsthat cause oncogenic activation of Ras lead to accumulation of Ras boundto GTP, the active form of the molecule. These mutations block theability of GAP to convert Ras to the inactive form. Mutations thatimpair the interactions of Ras with GAP also block the biologicalfunction of Ras.

While a number of Ras alleles exist (N-Ras, KRAS, H-Ras) which have beenimplicated in carcinogenesis, the type most often associated with colonand pancreatic carcinomas is KRAS. Nucleic acid molecules which aretargeted to certain regions of the KRAS allelic mRNAs may also proveinhibitory to the function of the other allelic mRNAs of the N-Ras andH-Ras genes.

The use of DsiRNA agents targeting KRAS therefore provides a class ofnovel therapeutic agents that can be used in the treatment, alleviation,or prevention of cancer and/or proliferative diseases, conditions, ordisorders, alone or in combination with other therapies.

Known human KRAS gene and polypeptide sequences include the following:

Wild-type KRAS sequence (SEQ ID NO: 1; K-Ras4A—transcript variant a;GenBank Accession No. NM_(—)033360.2):

GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGCACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCG ACACAGCAGGTCA AGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAGAGAGTGGAGGATGCTTTTTATACATTGGTGAGGGAGATCCGACAATACAGATTGAAAAAAATCAGCAAAGAAGAAAAGACTCCTGGCTGTGTGAAAATTAAAAAATGCATTATAATGTAATCTGGGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATGAGCAAAGATGGTAAAAAGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGTAAATACAATTTGTACTTTTTTCTTAAGGCATACTAGTACAAGTGGTAATTTTTGTACATTACACTAAATTATTTAGCATTTGTTTTAGCATTACCTAATTTTTTTCCTGCTCCATGCAGACTGTTAGCTTTTACCTTAAATGCTTATTTTAAAATGACAGTGGAAGTTTTTTTTTCCTCTAAGTGCCAGTATTCCCAGAGTTTTGGTTTTTGAACTAGCAATGCCTGTGAAAAAGAAACTGAATACCTAAGATTTCTGTCTTGGGGTTTTTGGTGCATGCAGTTGATTACTTCTTATTTTTCTTACCAATTGTGAATGTTGGTGTGAAACAAATTAATGAAGCTTTTGAATCATCCCTATTCTGTGTTTTATCTAGTCACATAAATGGATTAATTACTAATTTCAGTTGAGACCTTCTAATTGGTTTTTACTGAAACATTGAGGGAACACAAATTTATGGGCTTCCTGATGATGATTCTTCTAGGCATCATGTCCTATAGTTTGTCATCCCTGATGAATGTAAAGTTACACTGTTCACAAAGGTTTTGTCTCCTTTCCACTGCTATTAGTCATGGTCACTCTCCCCAAAATATTATATTTTTTCTATAAAAAGAAAAAAATGGAAAAAAATTACAAGGCAATGGAAACTATTATAAGGCCATTTCCTTTTCACATTAGATAAATTACTATAAAGACTCCTAATAGCTTTTCCTGTTAAGGCAACCCAGTGATGAAATGGGGATTATTATAGCAACCATTTTGGGGCTATATTTTACATGCTACTAAATTTTTATAATAATTGAAAAGATTTTAACAAGTATAAAAAATTCTCATAGGAATTAAATGTAGTCTCCCTGTGTCAGACTGCTCTTTCATAGTATAACTTTAAATCTTTTCTTCAACTTGAGTCTTTGAAGATAGTTTTAATTCTGCTTGTGACATTAAAAGATTATTTGGGCCAGTTATAGCTTATTAGGTGTTGAAGAGACCAAGGTTGCAAGGCCAGGCCCTGTGTGAACCTTTGAGCTTTCATAGAGAGTTTCACAGCATGGACTGTGTCCCCACGGTCATCCAGTGTTGTCATGCATTGGTTAGTCAAAATGGGGAGGGACTAGGGCAGTTTGGATAGCTCAACAAGATACAATCTCACTCTGTGGTGGTCCTGCTGACAAATCAAGAGCATTGCTTTTGTTTCTTAAGAAAACAAACTCTTTTTTAAAAATTACTTTTAAATATTAACTCAAAAGTTGAGATTTTGGGGTGGTGGTGTGCCAAGACATTAATTTTTTTTTTTAAACAATGAAGTGAAAAAGTTTTACAATCTCTAGGTTTGGCTAGTTCTCTTAACACTGGTTAAATTAACATTGCATAAACACTTTTCAAGTCTGATCCATATTTAATAATGCTTTAAAATAAAAATAAAAACAATCCTTTTGATAAATTTAAAATGTTACTTATTTTAAAATAAATGAAGTGAGATGGCATGGTGAGGTGAAAGTATCACTGGACTAGGAAGAAGGTGACTTAGGTTCTAGATAGGTGTCTTTTAGGACTCTGATTTTGAGGACATCACTTACTATCCATTTCTTCATGTTAAAAGAAGTCATCTCAAACTCTTAGTTTTTTTTTTTTACAACTATGTAATTTATATTCCATTTTACATAAGGATACACTTATTTGTCAAGCTCAGCACAATCTGTAAATTTTTAACCTATGTTACACCATCTTCAGTGCCAGTCTTGGGCAAAATTGTGCAAGAGGTGAAGTTTATATTTGAATATCCATTCTCGTTTTAGGACTCTTCTTCCATATTTAGTGTCATCTTGCCTCCCTACCTTCCACATGCCCCATGACTTGATGCAGTTTTAATACTTGTAATTCCCCTAACCATAAGATTTACTGCTGCTGTGGATATCTCCATGAAGTTTTCCCACTGAGTCACATCAGAAATGCCCTACATCTTATTTCCTCAGGGCTCAAGAGAATCTGACAGATACCATAAAGGGATTTGACCTAATCACTAATTTTCAGGTGGTGGCTGATGCTTTGAACATCTCTTTGCTGCCCAATCCATTAGCGACAGTAGGATTTTTCAAACCTGGTATGAATAGACAGAACCCTATCCAGTGGAAGGAGAATTTAATAAAGATAGTGCTGAAAGAATTCCTTAGGTAATCTATAACTAGGACTACTCCTGGTAACAGTAATACATTCCATTGTTTTAGTAACCAGAAATCTTCATGCAATGAAAAATACTTTAATTCATGAAGCTTACTTTTTTTTTTTGGTGTCAGAGTCTCGCTCTTGTCACCCAGGCTGGAATGCAGTGGCGCCATCTCAGCTCACTGCAACCTCCATCTCCCAGGTTCAAGCGATTCTCGTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGCGTGTGCCACTACACTCAACTAATTTTTGTATTTTTAGGAGAGACGGGGTTTCACCCTGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAAGTGATTCACCCACCTTGGCCTCATAAACCTGTTTTGCAGAACTCATTTATTCAGCAAATATTTATTGAGTGCCTACCAGATGCCAGTCACCGCACAAGGCACTGGGTATATGGTATCCCCAAACAAGAGACATAATCCCGGTCCTTAGGTAGTGCTAGTGTGGTCTGTAATATCTTACTAAGGCCTTTGGTATACGACCCAGAGATAACACGATGCGTATTTTAGTTTTGCAAAGAAGGGGTTTGGTCTCTGTGCCAGCTCTATAATTGTTTTGCTACGATTCCACTGAAACTCTTCGATCAAGCTACTTTATGTAAATCACTTCATTGTTTTAAAGGAATAAACTTGATTATATTGTTTTTTTATTTGGCATAACTGTGATTCTTTTAGGACAATTACTGTACACATTAAGGTGTATGTCAGATATTCATATTGACCCAAATGTGTAATATTCCAGTTTTCTCTGCATAAGTAATTAAAATATACTTAAAAATTAATAGTTTTATCTGGGTACAAATAAACAGGTGCCTGAACTAGTTCACAGACAAGGAAACTTCTATGTAAAAATCACTATGATTTCTGAATTGCTATGTGAAACTACAGATCTTTGGAACACTGTTTAGGTAGGGTGTTAAGACTTACACAGTACCTCGTTTCTACACAGAGAAAGAAATGGCCATACTTCAGGAACTGCAGTGCTTATGAGGGGATATTTAGGCCTCTTGAATTTTTGATGTAGATGGGCATTTTTTTAAGGTAGTGGTTAATTACCTTTATGTGAACTTTGAATGGTTTAACAAAAGATTTGTTTTTGTAGAGATTTTAAAGGGGGAGAATTCTAGAAATAAATGTTACCTAATTATTACAGCCTTAAAGACAAAAATCCTTGTTGAAGTTTTTTTAAAAAAAGCTAAATTACATAGACTTAGGCATTAACATGTTTGTGGAAGAATATAGCAGACGTATATTGTATCATTTGAGTGAATGTTCCCAAGTAGGCATTCTAGGCTCTATTTAACTGAGTCACACTGCATAGGAATTTAGAACCTAACTTTTATAGGTTATCAAAACTGTTGTCACCATTGCACAATTTTGTCCTAATATATACATAGAAACTTTGTGGGGCATGTTAAGTTACAGTTTGCACAAGTTCATCTCATTTGTATTCCATTGATTTTTTTTTTCTTCTAAACATTTTTTCTTCAAACAGTATATAACTTTTTTTAGGGGATTTTTTTTTAGACAGCAAAAACTATCTGAAGATTTCCATTTGTCAAAAAGTAATGATTTCTTGATAATTGTGTAGTAATGTTTTTTAGAACCCAGCAGTTACCTTAAAGCTGAATTTATATTTAGTAACTTCTGTGTTAATACTGGATAGCATGAATTCTGCATTGAGAAACTGAATAGCTGTCATAAAATGAAACTTTCTTTCTAAAGAAAGATACTCACATGAGTTCTTGAAGAATAGTCATAACTAGATTAAGATCTGTGTTTTAGTTTAATAGTTTGAAGTGCCTGTTTGGGATAATGATAGGTAATTTAGATGAATTTAGGGGAAAAAAAAGTTATCTGCAGATATGTTGAGGGCCCATCTCTCCCCCCACACCCCCACAGAGCTAACTGGGTTACAGTGTTTTATCCGAAAGTTTCCAATTCCACTGTCTTGTGTTTTCATGTTGAAAATACTTTTGCATTTTTCCTTTGAGTGCCAATTTCTTACTAGTACTATTTCTTAATGTAACATGTTTACCTGGAATGTATTTTAACTATTTTTGTATAGTGTAAACTGAAACATGCACATTTTGTACATTGTGCTTTCTTTTGTGGGACATATGCAGTGTGATCCAGTTGTTTTCCATCATTTGGTTGCGCTGACCTAGGAATGTTGGTCATATCAAACATTAAAAATGACCACTCTTTTAATTGAAATTAACTTTTAAATGTTTATAGGAGTATGTGCTGTGAAGTGATCTAAAATTTGTAATATTTTTGTCATGAACTGTACTACTCCTAATTATTGTAATGTAATAAAAATAGTTACAGTGACAAAAAAAAAAAAAAAThe underlined sequences above correspond to KRAS RNA sequences targetedby exemplified KRAS-355 and KRAS-940 DsiRNA agents of the invention.Known SNPs within the above cDNA sequence include an A/T polymorphism atposition 364 (dbSNP Accession No. rs17851045); a T/C polymorphism atposition 824 (dbSNP Accession No. rs1137282); and a KRAS G12V mutant G/Tpolymorphism at position 216, as previously described in US2005/0176045. These three polymorphic sites are shown in bold italics.Wild-type KRAS Amino Acid Sequence NP_(—)203524.1 (SEQ ID NO: 2;translation of NM_(—)033360):

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIMWild-type KRAS sequence (SEQ ID NO: 3; K-Ras4b—transcript variant b;GenBank Accession No. NM_(—)004985.3):

GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGCACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCG ACACAGCAGGTCA AGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAGGGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATGAGCAAAGATGGTAAAAAGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGTAAATACAATTTGTACTTTTTTCTTAAGGCATACTAGTACAAGTGGTAATTTTTGTACATTACACTAAATTATTAGCATTTGTTTTAGCATTACCTAATTTTTTTCCTGCTCCATGCAGACTGTTAGCTTTTACCTTAAATGCTTATTTTAAAATGACAGTGGAAGTTTTTTTTTCCTCTAAGTGCCAGTATTCCCAGAGTTTTGGTTTTTGAACTAGCAATGCCTGTGAAAAAGAAACTGAATACCTAAGATTTCTGTCTTGGGGTTTTTGGTGCATGCAGTTGATTACTTCTTATTTTTCTTACCAATTGTGAATGTTGGTGTGAAACAAATTAATGAAGCTTTTGAATCATCCCTATTCTGTGTTTTATCTAGTCACATAAATGGATTAATTACTAATTTCAGTTGAGACCTTCTAATTGGTTTTTACTGAAACATTGAGGGAACACAAATTTATGGGCTTCCTGATGATGATTCTTCTAGGCATCATGTCCTATAGTTTGTCATCCCTGATGAATGTAAAGTTACACTGTTCACAAAGGTTTTGTCTCCTTTCCACTGCTATTAGTCATGGTCACTCTCCCCAAAATATTATATTTTTTCTATAAAAAGAAAAAAATGGAAAAAAATTACAAGGCAATGGAAACTATTATAAGGCCATTTCCTTTTCACATTAGATAAATTACTATAAAGACTCCTAATAGCTTTTCCTGTTAAGGCAGACCCAGTATGAAATGGGGATTATTATAGCAACCATTTTGGGGCTATATTTACATGCTACTAAATTTTTATAATAATTGAAAAGATTTTAACAAGTATAAAAAATTCTCATAGGAATTAAATGTAGTCTCCCTGTGTCAGACTGCTCTTTCATAGTATAACTTTAAATCTTTTCTTCAACTTGAGTCTTTGAAGATAGTTTTAATTCTGCTTGTGACATTAAAAGATTATTTGGGCCAGTTATAGCTTATTAGGTGTTGAAGAGACCAAGGTTGCAAGGCCAGGCCCTGTGTGAACCTTTGAGCTTTCATAGAGAGTTTCACAGCATGGACTGTGTCCCCACGGTCATCCAGTGTTGTCATGCATTGGTTAGTCAAAATGGGGAGGGACTAGGGCAGTTTGGATAGCTCAACAAGATACAATCTCACTCTGTGGTGGTCCTGCTGACAAATCAAGAGCATTGCTTTTGTTTCTTAAGAAAACAAACTCTTTTTTAAAAATTACTTTTAAATATTAACTCAAAAGTTGAGATTTTGGGGTGGTGGTGTGCCAAGACATTAATTTTTTTTTTAAACAATGAAGTGAAAAAGTTTTACAATCTCTAGGTTTGGCTAGTTCTCTTAACACTGGTTAAATTAACATTGCATAAACACTTTTCAAGTCTGATCCATATTTAATAATGCTTTAAAATAAAAATAAAAACAATCCTTTTGATAAATTTAAAATGTTACTTATTTTAAAATAAATGAAGTGAGATGGCATGGTGAGGTGAAAGTATCACTGGACTAGGAAGAAGGTGACTTAGGTTCTAGATAGGTGTCTTTTAGGACTCTGATTTTGAGGACATCACTTACTATCCATTTCTTCATGTTAAAAGAAGTCATCTCAAACTCTTAGTTTTTTTTTTTTACAACTATGTAATTTATATTCCATTTACATAAGGATACACTTATTTGTCAAGCTCAGCACAATCTGTAAATTTTTAACCTATGTTACACCATCTTCAGTGCCAGTCTTGGGCAAAATTGTGCAAGAGGTGAAGTTTATATTTGAATATCCATTCTCGTTTTAGGACTCTTCTTCCATATTAGTGTCATCTTGCCTCCCTACCTTCCACATGCCCCATGACTTGATGCAGTTTTAATACTTGTAATTCCCCTAACCATAAGATTTACTGCTGCTGTGGATATCTCCATGAAGTTTTCCCACTGAGTCACATCAGAAATGCCCTACATCTTATTTCCTCAGGGCTCAAGAGAATCTGACAGATACCATAAAGGGATTTGACCTAATCACTAATTTTCAGGTGGTGGCTGATGCTTTGAACATCTCTTTGCTGCCCAATCCATTAGCGAAGTAGGATTTTTCAAACCTGGTATGAATAGACAGAACCCTATCCAGTGGAAGGAGAATTTAATAAAGATAGTGCTGAAAGAATTCCTTAGGTAATCTATAACTAGGACTACTCCTGGTAACAGTAATACATTCCATTGTTTTAGTAACCAGAAATCTTCATGCAATGAAAAATACTTTAATTCATGAAGCTTACTTTTTTTTTTTGGTGTCAGAGTCTCGCTCTTGTCACCCAGGCTGGAATGCAGTGGCGCCATCTCAGCTCACTGCAACCTCCATCTCCCAGGTTCAAGCGATTCTCGTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGCGTGTGCCACTACACTCAACTAATTTTTGTATTTTTAGGAGAGACGGGGTTTCACCCTGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAAGTGATTCACCCACCTTGGCCTCATAAACCTGTTTTGCAGAACTCATTTATTCAGCAAATATTTATTGAGTGCCTACCAGATGCCAGTCACCGCACAAGGCACTGGGTATATGGTATCCCCAAACAAGAGACATAATCCCGGTCCTTAGGTAGTGCTAGTGTGGTCTGTAATATCTTACTAAGGCCTTTGGTATACGACCCAGAGATAACACGATGCGTATTTTAGTTTTGCAAAGAAGGGGTTTGGTCTCTGTGCCAGCTCTATAATTGTTTTGCTACGATTCCACTGAAACTCTTCGATCAAGCTACTTTATGTAAATCACTTCATTGTTTTAAAGGAATAAACTTGATTATATTGTTTTTTTATTTGGCATAACTGTGATTCTTTTAGGACAATTACTGTACACATTAAGGTGTATGTCAGATATTCATATTGACCCAAATGTGTAATATTCCAGTTTTCTCTGCATAAGTAATTAAAATATACTTAAAAATTAATAGTTTTATCTGGGTACAAATAAACAGGTGCCTGAACTAGTTCACAGACAAGGAAACTTCTATGTAAAAATCACTATGATTTCTGAATTGCTATGTGAAACTACAGATCTTTGGAACACTGTTTAGGTAGGGTGTTAAGACTTACACAGTACCTCGTTTCTACACAGAGAAAGAAATGGCCATACTTCAGGAACTGCAGTGCTTATGAGGGGATATTTAGGCCTCTTGAATTTTTGATGTAGATGGGCATTTTTTTAAGGTAGTGGTTAATTACCTTTATGTGAACTTTGAATGGTTTAACAAAAGATTTGTTTTTGTAGAGATTTTAAAGGGGGAGAATTCTAGAAATAAATGTTACCTAATTATTACAGCCTTAAAGACAAAAATCCTTGTTGAAGTTTTTTTAAAAAAAGCTAAATTACATAGACTTAGGCATTAACATGTTTGTGGAAGAATATAGCAGACGTATATTGTATCATTTGAGTGAATGTTCCCAAGTAGGCATTCTAGGCTCTATTTAACTGAGTCACACTGCATAGGAATTTAGAACCTAACTTTTATAGGTTATCAAAACTGTTGTCACCATTGCACAATTTTGTCCTAATATATACATAGAAACTTTGTGGGGCATGTTAAGTTACAGTTTGCACAAGTTCATCTCATTTGTATTCCATTGATTTTTTTTTTCTTCTAAACATTTTTTCTTCAAACAGTATATAACTTTTTTTAGGGGATTTTTTTTTAGACAGCAAAAACTATCTGAAGATTTCCATTTGTCAAAAAGTAATGATTTCTTGATAATTGTGTAGTAATGTTTTTTAGAACCCAGCAGTTACCTTAAAGCTGAATTTATATTTAGTAACTTCTGTGTTAATACTGGATAGCATGAATTCTGCATTGAGAAACTGAATAGCTGTCATAAAATGAAACTTTCTTTCTAAAGAAAGATACTCACATGAGTTCTTGAAGAATAGTCATAACTAGATTAAGATCTGTGTTTTAGTTTAATAGTTTGAAGTGCCTGTTTGGGATAATGATAGGTAATTTAGATGAATTTAGGGGAAAAAAAAGTTATCTGCAGATATGTTGAGGGCCCATCTCTCCCCCCACACCCCCACAGAGCTAACTGGGTTACAGTGTTTTATCCGAAAGTTTCCAATTCCACTGTCTTGTGTTTTCATGTTGAAAATACTTTTGCATTTTTCCTTTGAGTGCCAATTTCTTACTAGTACTATTTCTTAATGTAACATGTTTACCTGGAATGTATTTTAACTATTTTTGTATAGTGTAAACTGAAACATGCACATTTTGTACATTGTGCTTTCTTTTGTGGGACATATGCAGTGTGATCCAGTTGTTTTCCATCATTTGGTTGCGCTGACCTAGGAATGTTGGTCATATCAAACATTAAAAATGACCACTCTTTTAATTGAAATTAACTTTTAAATGTTTATAGGAGTATGTGCTGTGAAGTGATCTAAAATTTGTAATATTTTTGTCATGAACTGTACTACTCCTAATTATTGTAATGTAATAAAAATAGTTACAGTGACAAAA AAAAAAAAAAAThe underlined sequences above correspond to KRAS RNA sequences targetedby exemplified KRAS-355 and KRAS-940 DsiRNA agents of the invention.Known SNPs within the above cDNA sequence include an A/T polymorphism atposition 364 (dbSNP Accession No. rs17851045); a T/C polymorphism atposition 700 (dbSNP Accession No. rs1137282); and a KRAS G12V mutant G/Tpolymorphism at position 216, as previously described in US2005/0176045. These three polymorphic sites are shown in bold italics.Wild-type KRAS Amino Acid Sequence NP_(—)004976.2 (SEQ ID NO: 4;translation of NM_(—)004985):

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIMIn Vitro Assay to Assess DsiRNA KRAS Inhibitory Activity

An in vitro assay that recapitulates RNAi in a cell-free system can beused to evaluate DsiRNA constructs targeting KRAS RNA sequence(s), andthus to assess KRAS-specific gene inhibitory activity (also referred toherein as KRAS inhibitory activity) of a DsiRNA. The assay comprises thesystem described by Tuschl et al., 1999, Genes and Development, 13,3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use withDsiRNA agents directed against KRAS RNA. A Drosophila extract derivedfrom syncytial blastoderm is used to reconstitute RNAi activity invitro. Target RNA is generated via in vitro transcription from anappropriate KRAS expressing plasmid using T7 RNA polymerase or viachemical synthesis. Sense and antisense DsiRNA strands (for example 20uM each) are annealed by incubation in buffer (such as 100 mM potassiumacetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minuteat 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer(for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mMmagnesium acetate). Annealing can be monitored by gel electrophoresis onan agarose gel in TBE buffer and stained with ethidium bromide. TheDrosophila lysate is prepared using zero to two-hour-old embryos fromOregon R flies collected on yeasted molasses agar that are dechorionatedand lysed. The lysate is centrifuged and the supernatant isolated. Theassay comprises a reaction mixture containing 50% lysate [vol/vol], RNA(10-50 pM final concentration), and 10% [vol/vol] lysis buffercontaining DsiRNA (10 nM final concentration). The reaction mixture alsocontains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100mM GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin(Promega), and 100 uM of each amino acid. The final concentration ofpotassium acetate is adjusted to 100 mM. The reactions are pre-assembledon ice and preincubated at 25° C. for 10 minutes before adding RNA, thenincubated at 25° C. for an additional 60 minutes. Reactions are quenchedwith 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNAcleavage is assayed by RT-PCR analysis or other methods known in the artand are compared to control reactions in which DsiRNA is omitted fromthe reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without DsiRNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in theKRAS RNA target for DsiRNA mediated RNAi cleavage, wherein a pluralityof DsiRNA constructs are screened for RNAi mediated cleavage of the KRASRNA target, for example, by analyzing the assay reaction byelectrophoresis of labeled target RNA, or by northern blotting, as wellas by other methodology well known in the art.

In certain embodiments, a DsiRNA of the invention is deemed to possessKRAS inhibitory activity if, e.g., a 50% reduction in KRAS RNA levels isobserved in a system, cell, tissue or organism, relative to a suitablecontrol. Additional metes and bounds for determination of KRASinhibitory activity of a DsiRNA of the invention are described supra.

Conjugation and Delivery of Anti-KRAS DsiRNA Agents

In certain embodiments the present invention relates to a method fortreating a subject having a KRAS-associated disease or disorder, or atrisk of developing a KRAS-associated disease or disorder. In suchembodiments, the DsiRNA can act as novel therapeutic agents forcontrolling the KRAS-associated disease or disorder. The methodcomprises administering a pharmaceutical composition of the invention tothe patient (e.g., human), such that the expression, level and/oractivity of a KRAS RNA is reduced. The expression, level and/or activityof a polypeptide encoded by a KRAS RNA might also be reduced by a DsiRNAof the instant invention, even where said DsiRNA is directed against anon-coding region of the KRAS transcript (e.g., a targeted 5′ UTR or 3′UTR sequence). Because of their high specificity, the DsiRNAs of thepresent invention can specifically target KRAS sequences of cells andtissues, optionally in an allele-specific manner where polymorphicalleles exist within an individual and/or population.

In the treatment of a KRAS-associated disease or disorder, the DsiRNAcan be brought into contact with the cells or tissue of a subject, e.g.,the cells or tissue of a subject exhibiting disregulation of KRAS and/orotherwise targeted for reduction of KRAS levels. For example, DsiRNAsubstantially identical to all or part of a KRAS RNA sequence, may bebrought into contact with or introduced into such a cell, either in vivoor in vitro. Similarly, DsiRNA substantially identical to all or part ofa KRAS RNA sequence may administered directly to a subject having or atrisk of developing a KRAS-associated disease or disorder.

Therapeutic use of the DsiRNA agents of the instant invention caninvolve use of formulations of DsiRNA agents comprising multipledifferent DsiRNA agent sequences. For example, two or more, three ormore, four or more, five or more, etc. of the presently described agentscan be combined to produce a formulation that, e.g., targets multipledifferent regions of the KRAS RNA, or that not only target KRAS RNA butalso target, e.g., cellular target genes associated with aKRAS-associated disease or disorder. A DsiRNA agent of the instantinvention may also be constructed such that either strand of the DsiRNAagent independently targets two or more regions of KRAS RNA, or suchthat one of the strands of the DsiRNA agent targets a cellular targetgene of KRAS known in the art.

Use of multifunctional DsiRNA molecules that target more then one regionof a target nucleic acid molecule can also provide potent inhibition ofKRAS RNA levels and expression. For example, a single multifunctionalDsiRNA construct of the invention can target both the KRAS-355 andKRAS-940 sites simultaneously; additionally and/or alternatively, singleor multifunctional agents of the invention can be designed toselectively target one splice variant of KRAS over another.

Thus, the DsiRNA agents of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treat,inhibit, reduce, or prevent a KRAS-associated disease or disorder. Forexample, the DsiRNA molecules can be administered to a subject or can beadministered to other appropriate cells evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

The DsiRNA molecules also can be used in combination with other knowntreatments to treat, inhibit, reduce, or prevent a KRAS-associateddisease or disorder in a subject or organism. For example, the describedmolecules could be used in combination with one or more known compounds,treatments, or procedures to treat, inhibit, reduce, or prevent aKRAS-associated disease or disorder in a subject or organism as areknown in the art.

A DsiRNA agent of the invention can be conjugated (e.g., at its 5′ or 3′terminus of its sense or antisense strand) or unconjugated to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye, cholesterol, or the like). Modifying DsiRNAagents in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting DsiRNA agent derivative ascompared to the corresponding unconjugated DsiRNA agent, are useful fortracing the DsiRNA agent derivative in the cell, or improve thestability of the DsiRNA agent derivative compared to the correspondingunconjugated DsiRNA agent.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

DsiRNA agents of the invention may be directly introduced into a cell(i.e., intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the nucleic acid. Vascular or extravascular circulation, theblood or lymph system, and the cerebrospinal fluid are sites where thenucleic acid may be introduced.

The DsiRNA agents of the invention can be introduced using nucleic aciddelivery methods known in art including injection of a solutioncontaining the nucleic acid, bombardment by particles covered by thenucleic acid, soaking the cell or organism in a solution of the nucleicacid, or electroporation of cell membranes in the presence of thenucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or otherwiseincrease inhibition of the target KRAS RNA.

A cell having a target KRAS RNA may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target KRAS RNA sequence and the dose ofDsiRNA agent material delivered, this process may provide partial orcomplete loss of function for the KRAS RNA. A reduction or loss of RNAlevels or expression (either KRAS RNA expression or encoded polypeptideexpression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of KRAS RNA levels or expressionrefers to the absence (or observable decrease) in the level of KRAS RNAor KRAS RNA-encoded protein. Specificity refers to the ability toinhibit the KRAS RNA without manifest effects on other genes of thecell. The consequences of inhibition can be confirmed by examination ofthe outward properties of the cell or organism or by biochemicaltechniques such as RNA solution hybridization, nuclease protection,Northern hybridization, reverse transcription, gene expressionmonitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS).Inhibition of target KRAS RNA sequence(s) by the DsiRNA agents of theinvention also can be measured based upon the effect of administrationof such DsiRNA agents upon development/progression of a KRAS-associateddisease or disorder, e.g., tumor formation, growth, metastasis, etc.,either in vivo or in vitro. Treatment and/or reductions in tumor orcancer cell levels can include halting or reduction of growth of tumoror cancer cell levels or reductions of, e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 99% or more, and can also be measured inlogarithmic terms, e.g., 10-fold, 100-fold, 1000-fold, 10⁵-fold,10⁶-fold, 10⁷-fold reduction in cancer cell levels could be achieved viaadministration of the DsiRNA agents of the invention to cells, a tissue,or a subject.

For RNA-mediated inhibition in a cell line or whole organism, expressiona reporter or drug resistance gene whose protein product is easilyassayed can be measured. Such reporter genes include acetohydroxyacidsynthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ),beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentarnycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracyclin. Depending on the assay, quantitation of theamount of gene expression allows one to determine a degree of inhibitionwhich is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to acell not treated according to the present invention.

Lower doses of injected material and longer times after administrationof RNA silencing agent may result in inhibition in a smaller fraction ofcells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targetedcells). Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target KRAS RNA ortranslation of target protein. As an example, the efficiency ofinhibition may be determined by assessing the amount of gene product inthe cell; RNA may be detected with a hybridization probe having anucleotide sequence outside the region used for the inhibitory DsiRNA,or translated polypeptide may be detected with an antibody raisedagainst the polypeptide sequence of that region.

The DsiRNA agent may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for apharmaceutical composition comprising the DsiRNA agent of the presentinvention. The DsiRNA agent sample can be suitably formulated andintroduced into the environment of the cell by any means that allows fora sufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsRNA are known inthe art and can be used so long as the dsRNA gains entry to the targetcells so that it can act. See, e.g., U.S. published patent applicationNos. 2004/0203145 A1 and 2005/0054598 A1. For example, the DsiRNA agentof the instant invention can be formulated in buffer solutions such asphosphate buffered saline solutions, liposomes, micellar structures, andcapsids. Formulations of DsiRNA agent with cationic lipids can be usedto facilitate transfection of the DsiRNA agent into cells. For example,cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(published PCT International Application WO 97/30731), can be used.Suitable lipids include Oligofectamine, Lipofectamine (LifeTechnologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.),or FuGene 6 (Roche) all of which can be used according to themanufacturer's instructions.

Such compositions typically include the nucleic acid molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The compounds can also be administered by transfection or infectionusing methods known in the art, including but not limited to the methodsdescribed in McCaffrey et al. (2002), Nature, 418(6893), 38-9(hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The compounds can also be administered by a method suitable foradministration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a nucleic acidmolecule (i.e., an effective dosage) depends on the nucleic acidselected. For instance, if a plasmid encoding a DsiRNA agent isselected, single dose amounts in the range of approximately 1 pg to 1000mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or1000 mg may be administered. In some embodiments, 1-5 g of thecompositions can be administered. The compositions can be administeredone from one or more times per day to one or more times per week;including once every other day. The skilled artisan will appreciate thatcertain factors may influence the dosage and timing required toeffectively treat a subject, including but not limited to the severityof the disease or disorder, previous treatments, the general healthand/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of aprotein, polypeptide, or antibody can include a single treatment or,preferably, can include a series of treatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The expression constructs may be constructs suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, e.g., Tuschl (2002, Nature Biotechnol 20:500-505).

It can be appreciated that the method of introducing DsiRNA agents intothe environment of the cell will depend on the type of cell and the makeup of its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the DsiRNA agents can be added directly to theliquid environment of the cells. Lipid formulations can also beadministered to animals such as by intravenous, intramuscular, orintraperitoneal injection, or orally or by inhalation or other methodsas are known in the art. When the formulation is suitable foradministration into animals such as mammals and more specificallyhumans, the formulation is also pharmaceutically acceptable.Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate DsiRNA agents in a buffer or saline solution anddirectly inject the formulated DsiRNA agents into cells, as in studieswith oocytes. The direct injection of DsiRNA agents duplexes may also bedone. For suitable methods of introducing dsRNA (e.g., DsiRNA agents),see U.S. published patent application No. 2004/0203145 A1.

Suitable amounts of a DsiRNA agent must be introduced and these amountscan be empirically determined using standard methods. Typically,effective concentrations of individual DsiRNA agent species in theenvironment of a cell will be 50 nanomolar or less, 10 nanomolar orless, or compositions in which concentrations of 1 nanomolar or less canbe used. In another embodiment, methods utilizing a concentration of 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, and even a concentration of 10 picomolar or less, 5picomolar or less, 2 picomolar or less or 1 picomolar or less can beused in many circumstances.

The method can be carried out by addition of the DsiRNA agentcompositions to an extracellular matrix in which cells can live providedthat the DsiRNA agent composition is formulated so that a sufficientamount of the DsiRNA agent can enter the cell to exert its effect. Forexample, the method is amenable for use with cells present in a liquidsuch as a liquid culture or cell growth media, in tissue explants, or inwhole organisms, including animals, such as mammals and especiallyhumans.

The level or activity of a KRAS RNA can be determined by a suitablemethod now known in the art or that is later developed. It can beappreciated that the method used to measure a target RNA and/or theexpression of a target RNA can depend upon the nature of the target RNA.For example, where the target KRAS RNA sequence encodes a protein, theterm “expression” can refer to a protein or the KRAS RNA/transcriptderived from the KRAS gene (either genomic or of exogenous origin). Insuch instances the expression of the target KRAS RNA can be determinedby measuring the amount of KRAS RNA/transcript directly or by measuringthe amount of KRas protein. Protein can be measured in protein assayssuch as by staining or immunoblotting or, if the protein catalyzes areaction that can be measured, by measuring reaction rates. All suchmethods are known in the art and can be used. Where target KRAS RNAlevels are to be measured, art-recognized methods for detecting RNAlevels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targetingKRAS RNAs with the DsiRNA agents of the instant invention, it is alsoanticipated that measurement of the efficacy of a DsiRNA agent inreducing levels of KRAS RNA or protein in a subject, tissue, in cells,either in vitro or in vivo, or in cell extracts can also be used todetermine the extent of reduction of KRAS-associated phenotypes (e.g.,disease or disorders, e.g., cancer or tumor formation, growth,metastasis, spread, etc.). The above measurements can be made on cells,cell extracts, tissues, tissue extracts or other suitable sourcematerial.

The determination of whether the expression of a KRAS RNA has beenreduced can be by a suitable method that can reliably detect changes inRNA levels. Typically, the determination is made by introducing into theenvironment of a cell undigested DsiRNA such that at least a portion ofthat DsiRNA agent enters the cytoplasm, and then measuring the level ofthe target RNA. The same measurement is made on identical untreatedcells and the results obtained from each measurement are compared.

The DsiRNA agent can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a DsiRNA agent andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a DsiRNA agenteffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

Suitably formulated pharmaceutical compositions of this invention can beadministered by means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general, a suitable dosage unit of dsRNA will be in the range of0.001 to 0.25 milligrams per kilogram body weight of the recipient perday, or in the range of 0.01 to 20 micrograms per kilogram body weightper day, or in the range of 0.01 to 10 micrograms per kilogram bodyweight per day, or in the range of 0.10 to 5 micrograms per kilogrambody weight per day, or in the range of 0.1 to 2.5 micrograms perkilogram body weight per day. Pharmaceutical composition comprising thedsRNA can be administered once daily. However, the therapeutic agent mayalso be dosed in dosage units containing two, three, four, five, six ormore sub-doses administered at appropriate intervals throughout the day.In that case, the dsRNA contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage unit.The dosage unit can also be compounded for a single dose over severaldays, e.g., using a conventional sustained release formulation whichprovides sustained and consistent release of the dsRNA over a severalday period. Sustained release formulations are well known in the art. Inthis embodiment, the dosage unit contains a corresponding multiple ofthe daily dose. Regardless of the formulation, the pharmaceuticalcomposition must contain dsRNA in a quantity sufficient to inhibitexpression of the target gene in the animal or human being treated. Thecomposition can be compounded in such a way that the sum of the multipleunits of dsRNA together contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For acompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsRNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by KRAS (e.g., misregulationand/or elevation of KRAS transcript and/or KRas protein levels), ortreatable via selective targeting of KRAS.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a DsiRNA agent or vectoror transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above (including, e.g.,prevention of the commencement of transforming events within a subjectvia inhibition of KRAS expression), by administering to the subject atherapeutic agent (e.g., a DsiRNA agent or vector or transgene encodingsame). Subjects at risk for the disease can be identified by, forexample, one or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the detection of, e.g., cancer in a subject, or the manifestation ofsymptoms characteristic of the disease or disorder, such that thedisease or disorder is prevented or, alternatively, delayed in itsprogression.

Another aspect of the invention pertains to methods of treating subjectstherapeutically, i.e., altering the onset of symptoms of the disease ordisorder. These methods can be performed in vitro (e.g., by culturingthe cell with the DsiRNA agent) or, alternatively, in vivo (e.g., byadministering the DsiRNA agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target KRAS RNAmolecules of the present invention or target KRAS RNA modulatorsaccording to that individual's drug response genotype. Pharmacogenomicsallows a clinician or physician to target prophylactic or therapeutictreatments to patients who will most benefit from the treatment and toavoid treatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, a DsiRNA agent (or expression vector or transgene encodingsame) as described herein can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with said agent.Alternatively, an agent (e.g., a therapeutic agent) can be used in ananimal model to determine the mechanism of action of such an agent.

Models Useful to Evaluate the Down-Regulation of KRAS mRNA Levels andExpression

Cell Culture

Kita et al., 1999, Int. J. Cancer, 80, 553-558, describes the growthinhibition of human pancreatic cancer cell lines by antisenseoligonucleotides specific to mutated KRAS genes. Antisenseoligonucleotides were transfected to the transformed cells usingliposomes. Cellular proliferation, KRAS mRNA expression, and KRasprotein synthesis were all evaluated as endpoints. Sato et al., 2000,Cancer Lett., 155, 153-161, describes another human pancreatic cancercell line, HOR-P1, that is characterized by high angiogenic activity andmetastatic potential. Genetic and molecular analysis of this cell linerevealed both increased telomerase activity and a mutation in the KRASoncogene.

The DsiRNA agents of the invention can be tested for cleavage activityin vivo, for example, using the following procedure. The nucleotidesequences within the KRAS cDNA targeted by the DsiRNA agents of theinvention are shown in the above KRAS sequences.

The DsiRNA reagents of the invention can be tested in cell culture usingHeLa or other mammalian cells to determine the extent of KRAS RNA andKRas protein inhibition. DsiRNA reagents (e.g., see FIGS. 1 and 4, andabove-recited structures) are selected against the KRAS target asdescribed herein. KRAS RNA inhibition is measured after delivery ofthese reagents by a suitable transfection agent to, for example,cultured HeLa cells or other transformed or non-transformed mammaliancells in culture. Relative amounts of target KRAS RNA are measuredversus actin or other appropriate control using real-time PCR monitoringof amplification (e.g., ABI 7700 TAQMAN®). A comparison is made to amixture of oligonucleotide sequences made to unrelated targets or to arandomized DsiRNA control with the same overall length and chemistry,but randomly substituted at each position, or simply to appropriatevehicle-treated or untreated controls. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead DsiRNA molecule.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following DsiRNA delivery, for example,using Ambion Rnaqueous 4-PCR purification kit for large scaleextractions, or Ambion Rnaqueous-96 purification kit for 96-well assays.For Taqman analysis, dual-labeled probes are synthesized with, forexample, the reporter dyes FAM or VIC covalently linked at the 5′-endand the quencher dye TAMARA conjugated to the 3′-end. One-step RT-PCRamplifications are performed on, for example, an ABI PRISM 7700 Sequencedetector using 50 uL reactions consisting of 10 uL total RNA, 100 nMforward primer, 100 mM reverse primer, 100 nM probe, 1×TaqMan PCRreaction buffer (PE-Applied Biosystems), 5.5 mM MgCl2, 100 uM each dATP,dCTP, dGTP and dTTP, 0.2U RNase Inhibitor (Promega), 0.025U AmpliTaqGold (PE-Applied Biosystems) and 0.2U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of target KRAS mRNA level isdetermined relative to standards generated from serially diluted totalcellular RNA (300, 100, 30, 10 ng/r×n) and normalizing to, for example,36B4 mRNA in either parallel or same tube TaqMan reactions.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hours at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, DsiRNA molecules of the invention are complexed withcationic lipids for cell culture experiments. DsiRNA and cationic lipidmixtures are prepared in serum-free DMEM immediately prior to additionto the cells. DMEM plus additives are warmed to room temperature (about20-25° C.) and cationic lipid is added to the final desiredconcentration and the solution is vortexed briefly. DsiRNA molecules areadded to the final desired concentration and the solution is againvortexed briefly and incubated for 10 minutes at room temperature. Indose response experiments, the RNA/lipid complex is serially dilutedinto DMEM following the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-KRAS DsiRNA agents in animal models isan important prerequisite to human clinical trials. Various animalmodels of cancer and/or proliferative diseases, conditions, or disordersas are known in the art can be adapted for use for pre-clinicalevaluation of the efficacy of DsiRNA compositions of the invention inmodulating KRAS gene expression toward therapeutic use.

As in cell culture models, the most Ras sensitive mouse tumor xenograftsare those derived from cancer cells that express mutant Ras proteins.Nude mice bearing H-Ras transformed bladder cancer cell xenografts weresensitive to an anti-Ras antisense nucleic acid, resulting in an 80%inhibition of tumor growth after a 31 day treatment period (Wickstrom,2001, Mol. Biotechnol., 18, 35-35). Zhang et al., 2000, Gene Ther., 7,2041, describes an anti-KRAS ribozyme adenoviral vector (KRbz-ADV)targeting a KRAS mutant (KRAS codon 12 GGT to GTT; H441 and H1725 cellsrespectively). Non-small cell lung cancer cells (NSCLC H441 and H1725cells) that express the mutant KRas protein were used in nude mousexenografts compared to NSCLC H1650 cells that lack the relevantmutation. Pre-treatment with KRbz-ADV completely abrogated engraftmentof both H441 and H1725 cells and compared to 100% engraftment and tumorgrowth in animals that received untreated tumor cells or a controlvector. Additional mouse models of KRAS misregulation/mutation have alsobeen described (e.g., in Kim et al. Cell 121: 823-835, which identifieda role of KRAS in promoting lung adenocarcinomas). The above studiesprovide proof that inhibition of Ras expression (e.g., KRAS expression)by anti-Ras agents causes inhibition of tumor growth in animals.

As such, these models can be used in evaluating the efficacy of DsiRNAmolecules of the invention in inhibiting KRAS levels, expression,tumor/cancer formation, growth, spread, development of otherKRAS-associated phenotypes, diseases or disorders, etc. These models andothers can similarly be used to evaluate the safety/toxicity andefficacy of DsiRNA molecules of the invention in a pre-clinical setting.

Specific examples of animal model systems useful for evaluation of theKRAS-targeting DsiRNAs of the instant invention include wild-type mice,orthotopic HCC xenograft tumor model mice (e.g., Hep3B and HepG2) anddisseminated melanoma model mice. In an exemplary in vivo experiment,DsiRNAs of the invention are tail vein injected into such mouse modelsat doses ranging from 1 to 10 mg/kg or, alternatively, repeated dosesare administered at single-dose IC₅₀ levels, and organs (e.g., liver,kidney, lung, pancreas, colon, skin, spleen, bone marrow, lymph nodes,mammary fat pad, etc.) are harvested 24 hours after administration ofthe final dose. Such organs are then evaluated for mouse and/or humanKRAS levels, depending upon the model used. Duration of action can alsobe examined at, e.g., 1, 4, 7, 14, 21 or more days after final DsiRNAadministration.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Anti-KRAS DsiRNA Design

Preferred anti-KRAS DsiRNA agents were selected from a pre-screenedpopulation of DsiRNAs. Design of DsiRNAs can optionally involve use ofpredictive scoring algorithms that perform in silico assessments of theprojected activity/efficacy of a number of possible DsiRNAs spanning aregion of sequence.

Example 2 Preparation of Double-Stranded RNA Oligonucleotides

Oligonucleotide Synthesis and Purification

DsiRNA molecules can be designed to interact with various sites in theRNA message, for example, target sequences within the RNA sequencesdescribed herein. In presently exemplified agents, two target KRASsequences were selected. The sequence of one strand of the DsiRNAmolecules were complementary to the target KRAS site sequences describedabove. The DsiRNA molecules were chemically synthesized using methodsdescribed herein. Generally, DsiRNA constructs were synthesized usingsolid phase oligonucleotide synthesis methods as described for 19-23mersiRNAs (see for example Usman et al., U.S. Pat. Nos. 5,804,683;5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117;6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400;6,111,086).

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies, Coralville, Iowa). Forexample, RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques(Damha and Olgivie, 1993, Methods Mol Biol 20: 81-114; Wincott et al.,1995, Nucleic Acids Res 23: 2677-84). The oligomers were purified usingion-exchange high performance liquid chromatography (IE-HPLC) on anAmersham Source 15Q column (1.0 cm×25 cm; Amersham Pharmacia Biotech,Piscataway, N.J.) using a 15 min step-linear gradient. The gradientvaries from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Sampleswere monitored at 260 nm and peaks corresponding to the full-lengtholigonucleotide species are collected, pooled, desalted on NAP-5columns, and lyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).The CE capillaries had a 100 μm inner diameter and contains ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, run in an electric field of 444 V/cm anddetected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere obtained that are at least 90% pure as assessed by CE for use inexperiments described below. Compound identity was verified bymatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectroscopy on a Voyager DE™ Biospectometry Work Station (AppliedBiosystems, Foster City, Calif.) following the manufacturer'srecommended protocol. Relative molecular masses of all oligomers wereobtained, often within 0.2% of expected molecular mass.

Preparation of Duplexes

Single-stranded RNA (ssRNA) oligomers were resuspended, e.g., at 100 μMconcentration in duplex buffer consisting of 100 mM potassium acetate,30 mM HEPES, pH 7.5. Complementary sense and antisense strands weremixed in equal molar amounts to yield a final solution of, e.g., 50 μMduplex. Samples were heated to 100° C. for 5′ in RNA buffer (IDT) andallowed to cool to room temperature before use. Double-stranded RNA(dsRNA) oligomers were stored at −20° C. Single-stranded RNA oligomerswere stored lyophilized or in nuclease-free water at −80° C.

Nomenclature

For consistency, the following nomenclature has been employed in theinstant specification. Names given to duplexes indicate the length ofthe oligomers and the presence or absence of overhangs. A “25/27” is anasymmetric duplex having a 25 base sense strand and a 27 base antisensestrand with a 2-base 3′-overhang. A “27/25” is an asymmetric duplexhaving a 27 base sense strand and a 25 base antisense strand.

Cell Culture and RNA Transfection

HeLa cells were obtained from ATCC and maintained in Dulbecco's modifiedEagle medium (HyClone) supplemented with 10% fetal bovine serum(HyClone) at 37° C. under 5% CO₂. For RNA transfections, HeLa cells weretransfected with DsiRNAs as indicated at a final concentration of 1 nMor 0.1 nM using Lipofectamine™ RNAiMAX (Invitrogen) and followingmanufacturer's instructions. Briefly, 2.5 μL of a 0.2 μM or 0.02 μMstock solution of each DsiRNA were mixed with 46.5 μL of Opti-MEM I(Invitrogen) and 1 μL of Lipofectamine™ RNAiMAX. The resulting 50 μL mixwas added into individual wells of 12 well plates and incubated for 20min at RT to allow DsiRNA:Lipofectamine™ RNAiMAX complexes to form.Meanwhile, HeLa cells were trypsinized and resuspended in medium at afinal concentration of 367 cells/μL. Finally, 450 μL of the cellsuspension were added to each well (final volume 500 μL) and plates wereplaced into the incubator for 24 hours.

Assessment of KRAS Inhibition

KRAS target gene knockdown was determined by qRT-PCR, with valuesnormalized to HPRT expression control treatments, includingLipofectamine™ RNAiMAX alone (Vehicle control) or untreated.

RNA Isolation and Analysis

Cells were washed once with 2 mL of PBS, and total RNA was extractedusing RNeasy Mini Kit™ (Qiagen) and eluted in a final volume of 30 μL. 1μg of total RNA was reverse-transcribed using Transcriptor 1^(st) StrandcDNA Kit™ (Roche) and random hexamers following manufacturer'sinstructions. One-thirtieth (0.66 μL) of the resulting cDNA was mixedwith 5 μL of IQ Multiplex Powermix (Bio-Rad) together with 3.33 μL ofH₂O and 1 μL of a 3 μM mix containing primers and probes specific forhuman genes HPRT-1 (accession number NM_(—)000194) and KRAS targetsequences.

Quantitative RT-PCR

A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) was usedfor the amplification reactions. PCR conditions were: 95° C. for 3 min;and then cycling at 95° C., 10 sec; 55° C., 1 min for 40 cycles. Eachsample was tested in triplicate. Relative HPRT mRNA levels werenormalized to KRAS mRNA levels and compared with mRNA levels obtained incontrol samples treated with the transfection reagent alone, oruntreated. Data was analyzed using Bio-Rad CFX Manager version 1.0software.

Example 3 KRAS-355 Targeted DsiRNA Inhibition of KRAS

DsiRNA molecules targeting KRAS were designed and synthesized asdescribed above and tested in HeLa cells for inhibitory efficacy. Theability of DsiRNA agents possessing varying end structures but commonlydirected against the same KRAS cDNA target sequence(5′-AGCAGGTCAAGAGGAGTACAGTGCAAT-3′ (SEQ ID NO: 147)) to inhibit KRASexpression was assessed in comparison to corresponding KRAS targetsequence-directed 21mer siRNAs (refer to FIG. 1 for anti-KRAS agentstructures tested). To perform such experiments, HeLa cells were platedapproximately 24 hours before transfection in 96-well plates at5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells were 70-90% confluent. For transfection, annealedDsiRNAs were mixed with the transfection reagent (Lipofectamine™RNAiMAX, Invitrogen) in a volume of 50 μl/well and incubated for 20minutes at room temperature. The DsiRNA transfection mixtures were addedto cells to give a final DsiRNA concentration of 1 nM (FIG. 2) or 100 pM(FIG. 3) in a volume of 150 μl. Each DsiRNA transfection mixture wasadded to 3 wells for triplicate DsiRNA treatments. Cells were incubatedat 37° C. for 24 hours in the continued presence of the DsiRNAtransfection mixture. At 24 hours, RNA was prepared from each well oftreated cells. The supernatants with the transfection mixtures werefirst removed and discarded, then the cells were lysed and RNA preparedfrom each well. Target KRAS RNA levels following treatment wereevaluated by qRT-PCR for the KRAS target gene, with values normalized tothose obtained for a vehicle-treated control. Triplicate data wasaveraged and the standard deviations determined for each treatment.Normalized data were graphed and the fold reduction of target mRNA byactive DsiRNAs in comparison to siRNAs and vehicle or untreated controlswas determined.

As shown in FIG. 2, the tested 25/27mer DsiRNA agent (DP1148P/DP1151G)showed significantly greater KRAS inhibitory efficacy at 1 nMconcentration than an optimized 21 mer siRNA (DP1158P/DP1159G siRNA)directed against the same KRAS target sequence and sharing the sameprojected Ago2 cleavage site within the target KRAS transcript sequenceas all other agents of FIG. 1 that were tested (such Ago2 cleavage sitealignment normalizes for variations that might otherwise be attributableto varying levels of RISC activity). Notably, Dicer enzyme cleavage ofthe tested 25/27mer DsiRNA was projected to generate the exact same“optimized” 21mer siRNA as the “optimized” 21mer tested, with theresults obtained emphasizing that DsiRNA agents possess specialefficacy/potency advantages over corresponding siRNA agents. Similarlydramatic results at 1 nM concentration were observed for both 27merblunt/blunt and 27mer blunt/fray DsiRNA agents directed against theKRAS-355 sequence, as compared to 21mer siRNAs possessing blunt/bluntand blunt/fray end structures which were similarly directed against theKRAS-355 sequence. Thus, the 25/27mer and 27mer DsiRNAs testedreproducibly exhibited enhanced inhibitory efficacy against the targetedKRAS transcript as compared to the corresponding 21mer agentsconcurrently tested. Indeed, all 25/27mer and 27mer DsiRNAs tested at 1nM concentration surprisingly outperformed all 21mer siRNAs tested atthe same concentration.

Robust inhibitory efficacies were also observed for the anti-KRAS-355DsiRNA agents at 100 pM concentration (FIG. 3). The tested 25/27merDsiRNA agent (DP1148P/DP1151G) showed significantly greater KRASinhibitory efficacy at 100 pM concentration than the optimized 21mersiRNA (DP1158P/DP1159G siRNA) directed against the same KRAS-355 targetsequence. Similarly dramatic results at 100 pM concentration wereobserved for 27mer blunt/fray DsiRNA agents directed against theKRAS-355 sequence, as compared to the corresponding 21 mer siRNApossessing blunt/fray end structures. The 27mer blunt/blunt agent was atleast equally as effective as the corresponding 21 mer blunt/blunt agentthat was tested, even if the apparent enhancement of 27mer blunt/bluntagent efficacy relative to 21 mer blunt/blunt agent efficacy was notstatistically significant at 100 pM concentration. Thus, the 25/27merand 27mer blunt/fray DsiRNAs tested reproducibly exhibited enhancedinhibitory efficacy against the targeted KRAS transcript as compared tothe corresponding 21mer siRNA agents concurrently tested at 100 pM.

Example 4 KRAS-940 Targeted DsiRNA Inhibition of KRAS

DsiRNA molecules targeting KRAS were designed and synthesized asdescribed above and tested in HeLa cells for inhibitory efficacy asdescribed in Example 3. The ability of DsiRNA agents possessing varyingend structures but commonly directed against the same KRAS cDNA targetsequence (5′-TATTAGCATTTTGITTTAGCATTACCTA-3′ (SEQ ID NO: 179)) toinhibit KRAS expression was assessed in comparison to corresponding KRAStarget sequence-directed 21mer siRNAs (refer to FIG. 4 for anti-KRASagent structures tested). As shown in FIG. 5, the tested 25/27mer DsiRNAagent (DP1136P/DP1139G) showed significantly greater KRAS inhibitoryefficacy at 1 nM concentration than an optimized 21mer siRNA(DP1146P/DP1147G siRNA) directed against the same KRAS target sequenceand sharing the same projected Ago2 cleavage site within the target KRAStranscript sequence as all other agents of FIG. 4 that were tested (suchAgo2 cleavage site alignment normalizes for variations that mightotherwise be attributable to varying levels of RISC activity). Notably,Dicer enzyme cleavage of the tested 25/27mer DsiRNA was projected togenerate the exact same “optimized” 21mer siRNA as the “optimized” 21mertested, with the results obtained emphasizing that DsiRNA agents possessspecial efficacy/potency advantages over corresponding siRNA agents.Similarly dramatic results at 1 nM concentration were observed for both27mer blunt/blunt and 27mer blunt/fray DsiRNA agents directed againstthe KRAS-940 sequence, as compared to 21mer siRNAs possessingblunt/blunt and blunt/fray end structures which were similarly directedagainst the KRAS-940 sequence. Thus, the 25/27mer and 27mer DsiRNAstested reproducibly exhibited enhanced inhibitory efficacy against thetargeted KRAS transcript as compared to the corresponding 21mer agentsconcurrently tested. Indeed, all 25/27mer and 27mer DsiRNAs tested at 1nM concentration unexpectedly outperformed all 21 mer siRNAs tested atthe same concentration.

Robust inhibitory efficacies were also observed for the anti-KRAS-940DsiRNA agents at 100 pM concentration (FIG. 6). The tested 25/27merDsiRNA agent (DP1136P/DP1139G) showed significantly greater KRASinhibitory efficacy at 100 pM concentration than the optimized 21mersiRNA (DP1146P/DP1147G siRNA) directed against the same KRAS-940 targetsequence. Similarly dramatic results at 100 pM concentration wereobserved for 27mer blunt/blunt and blunt/fray DsiRNA agents directedagainst the KRAS-940 sequence, as compared to the corresponding 21mersiRNAs possessing blunt/blunt and blunt/fray end structures. Thus, the25/27mer and 27mer blunt/blunt and blunt/fray DsiRNAs testedreproducibly exhibited enhanced inhibitory efficacy against the targetedKRAS transcript as compared to the corresponding 21mer siRNA agentsconcurrently tested at 100 pM. Indeed, all anti-KRAS-940 25/27mer and27mer DsiRNAs tested at 100 pM concentration unexpectedly outperformedall 21mer siRNAs which were tested at the same concentration.

Example 5 Further DsiRNA Inhibition of KRAS

The forty DsiRNA molecules shown in Table 3 possessing antisense strandSEQ ID NOs: 11-50 and targeting KRAS wild-type sequences (the DsiRNAs ofTable 3 targeting alternative/polymorphic sequences were not tested)were designed and synthesized as described above and tested in HeLacells for inhibitory efficacy as described in Example 3 above. Theability of these DsiRNA agents to inhibit KRAS expression was assessedin comparison to corresponding KRAS target sequence-directed 21 mersiRNAs (refer to Table 2 for corresponding anti-KRAS 21mer agentantisense strands tested, and to FIG. 7 for a schematic representationof the experiment). All DsiRNA agents showed efficacy as KRASinhibitors, with 35 of 40 tested DsiRNA agents exhibiting greater than50% reduction of the KRAS target. As shown in FIG. 8, for four out ofevery five DsiRNA-cognate siRNA pairs tested, the DsiRNA agent exhibitedsignificantly superior efficacy in decreasing levels of KRAS target thanthe cognate siRNA agent. The duration of such inhibitory effects wasalso examined at both 24 hours and 48 hours post-administration, withconcentrations of 1 nM and 5 nM tested. For 26 of the 40 DsiRNA-cognatesiRNA pairs, the DsiRNA agent showed statistically significant enhancedlevels of KRAS target inhibition than the corresponding cognate siRNAagent at all concentrations and durations tested (FIG. 9). This resultwas in marked contrast to the only 6 of 40 instances in which thecognate siRNA agent outperformed the DsiRNA agent (FIG. 9). Thus,statistically significant distinctions were observed between DsiRNAs andmatched cognate siRNAs (possessing aligned projected Agog cleavagesites) across the KRAS target RNA. By a large majority, the DsiRNAsdramatically and unexpectedly outperformed cognate siRNAs. Importantly,these results demonstrated that DsiRNA activity did not directlycorrelate with siRNA activity, nor did the converse hold. Accordingly,the above results demonstrated that DsiRNAs and siRNAs engage the RNAinterference machinery differently, and that DsiRNAs and siRNAs—in spiteof both comprising double-stranded RNA—are, in fact, different drugs.

As shown in FIGS. 10 and 11, IC₅₀ values were determined for 29 of these40 KRAS-targeting asymmetric DsiRNAs. Remarkably, 15 of these 29asymmetric DsiRNAs exhibited IC₅₀ values below 10 pM, furtherdocumenting the remarkable potency of these DsiRNAs.

Example 6 Assay of 243 Selected KRAS-Targeting DsiRNAs for KRASInhibition

In this example, 243 asymmetric DsiRNAs (here, as above, tested DsiRNAspossessed a 25/27mer structure) were constructed and tested for KRASinhibitory efficacy in human HeLa and mouse Hepa 1-6 cells incubated inthe presence of such DsiRNAs at a concentration of 1 nM. The 243asymmetric DsiRNAs tested included a subset of DsiRNAs selected fromTables 2-5 above, as well as a further set of asymmetric DsiRNAsdesigned to target specific sequences within human KRAS, mouse KRAS, orboth. Sequences and structures of these additional 243 asymmetricDsiRNAs are shown in Table 8.

TABLE 8 Additional Anti-KRAS Asymmetric (25/27 mer) DsiRNA Agent Structures Tested in Human HeLa and Mouse  Hepa 1-6 Cells  5′-GAGGCCUGCUGAAAAUGACUGAAta-3′ (SEQ ID NO: 4650)3′-CUCUCCGGACGACUUUUACUGACUUAU-5′ (SEQ ID NO: 4407) KRAS-166 Target:5′-GAGAGGCCTGCTGAAAATGACTGAATA-3′ (SEQ ID NO: 4893)  5′-AGGCCUGCUGAAAAUGACUGAAUat-3′ (SEQ ID NO: 4651)3′-UCUCCGGACGACUUUUACUGACUUAUA-5′ (SEQ ID NO: 4408) KRAS-167 Target:5′-AGAGGCCTGCTGAAAATGACTGAATAT-3′ (SEQ ID NO: 4894)  5′-GGCCUGCUGAAAAUGACUGAAUAta-3′ (SEQ ID NO: 4652)3′-CUCCGGACGACUUUUACUGACUUAUAU-5′ (SEQ ID NO: 4409) KRAS-168 Target:5′-GAGGCCTGCTGAAAATGACTGAATATA-3′ (SEQ ID NO: 4895)  5′-GCCUGCUGAAAAUGACUGAAUAUaa-3′ (SEQ ID NO: 4653)3′-UCCGGACGACUUUUACUGACUUAUAUU-5′ (SEQ ID NO: 4410) KRAS-169 Target:5′-AGGCCTGCTGAAAATGACTGAATATAA-3′ (SEQ ID NO: 4896)  5′-GUUGGAGCUGGUGGCGUAGGCAAga-3′ (SEQ ID NO: 4654)3′-AUCAACCUCGACCACCGCAUCCGUUCU-5′ (SEQ ID NO: 4411) KRAS-204 Target:5′-TAGTTGGAGCTGGTGGCGTAGGCAAGA-3′ (SEQ ID NO: 4897)  5′-UUGGAGCUGGUGGCGUAGGCAAGag-3′ (SEQ ID NO: 4655)3′-UCAACCUCGACCACCGCAUCCGUUCUC-5′ (SEQ ID NO: 4412) KRAS-205 Target:5′-AGTTGGAGCTGGTGGCGTAGGCAAGAG-3′ (SEQ ID NO: 4898)  5′-UGGAGCUGGUGGCGUAGGCAAGAgt-3′ (SEQ ID NO: 4656)3′-CAACCUCGACCACCGCAUCCGUUCUCA-5′ (SEQ ID NO: 4413) KRAS-206 Target:5′-GTTGGAGCTGGTGGCGTAGGCAAGAGT-3′ (SEQ ID NO: 4899)  5′-GGAGCUGGUGGCGUAGGCAAGAGtg-3′ (SEQ ID NO: 4657)3′-AACCUCGACCACCGCAUCCGUUCUCAC-5′ (SEQ ID NO: 4414) KRAS-207 Target:5′-TTGGAGCTGGTGGCGTAGGCAAGAGTG-3′ (SEQ ID NO: 4900)  5′-GAGCUGGUGGCGUAGGCAAGAGUgc-3′ (SEQ ID NO: 4658)3′-ACCUCGACCACCGCAUCCGUUCUCACG-5′ (SEQ ID NO: 4415) KRAS-208 Target:5′-TGGAGCTGGTGGCGTAGGCAAGAGTGC-3′ (SEQ ID NO: 4901)  5′-AGCUGGUGGCGUAGGCAAGAGUGcc-3′ (SEQ ID NO: 4659)3′-CCUCGACCACCGCAUCCGUUCUCACGG-5′ (SEQ ID NO: 4416) KRAS-209 Target:5′-GGAGCTGGTGGCGTAGGCAAGAGTGCC-3′ (SEQ ID NO: 4902)  5′-GCUGGUGGCGUAGGCAAGAGUGCct-3′ (SEQ ID NO: 4660)3′-CUCGACCACCGCAUCCGUUCUCACGGA-5′ (SEQ ID NO: 4417) KRAS-210 Target:5′-GAGCTGGTGGCGTAGGCAAGAGTGCCT-3′ (SEQ ID NO: 4903)  5′-UACAGCUAAUUCAGAAUCAUUUUgt-3′ (SEQ ID NO: 4661)3′-CUAUGUCGAUUAAGUCUUAGUAAAACA-5′ (SEQ ID NO: 4418) KRAS-241 Target:5′-GATACAGCTAATTCAGAATCATTTTGT-3′ (SEQ ID NO: 4904)  5′-UAAUUGAUGGAGAAACCUGUCUCtt-3′ (SEQ ID NO: 4662)3′-UCAUUAACUACCUCUUUGGACAGAGAA-5′ (SEQ ID NO: 4419) KRAS-313 Target:5′-AGTAATTGATGGAGAAACCTGTCTCTT-3′ (SEQ ID NO: 4905)  5′-AAUUGAUGGAGAAACCUGUCUCUtg-3′ (SEQ ID NO: 4663)3′-CAUUAACUACCUCUUUGGACAGAGAAC-5′ (SEQ ID NO: 4420) KRAS-314 Target:5′-GTAATTGATGGAGAAACCTGTCTCTTG-3′ (SEQ ID NO: 4906)  5′-GAUGGAGAAACCUGUCUCUUGGAta-3′ (SEQ ID NO: 4664)3′-AACUACCUCUUUGGACAGAGAACCUAU-5′ (SEQ ID NO: 4421) KRAS-318 Target:5′-TTGATGGAGAAACCTGTCTCTTGGATA-3′ (SEQ ID NO: 4907)  5′-CCUGUCUCUUGGAUAUUCUCGACac-3′ (SEQ ID NO: 4665)3′-UUGGACAGAGAACCUAUAAGAGCUGUG-5′ (SEQ ID NO: 4422) KRAS-328 Target:5′-AACCTGTCTCTTGGATATTCTCGACAC-3′ (SEQ ID NO: 4908)  5′-UGUCUCUUGGAUAUUCUCGACACag-3′ (SEQ ID NO: 4666)3′-GGACAGAGAACCUAUAAGAGCUGUGUC-5′ (SEQ ID NO: 4423) KRAS-330 Target:5′-CCTGTCTCTTGGATATTCTCGACACAG-3′ (SEQ ID NO: 4909)  5′-GUCUCUUGGAUAUUCUCGACACAgc-3′ (SEQ ID NO: 4667)3′-GACAGAGAACCUAUAAGAGCUGUGUCG-5′ (SEQ ID NO: 4424) KRAS-331 Target:5′-CTGTCTCTTGGATATTCTCGACACAGC-3′ (SEQ ID NO: 4910)  5′-UCUCUUGGAUAUUCUCGACACAGca-3′ (SEQ ID NO: 4668)3′-ACAGAGAACCUAUAAGAGCUGUGUCGU-5′ (SEQ ID NO: 4425) KRAS-332 Target:5′-TGTCTCTTGGATATTCTCGACACAGCA-3′ (SEQ ID NO: 4911)  5′-CUCUUGGAUAUUCUCGACACAGCag-3′ (SEQ ID NO: 4669)3′-CAGAGAACCUAUAAGAGCUGUGUCGUC-5′ (SEQ ID NO: 4426) KRAS-333 Target:5′-GTCTCTTGGATATTCTCGACACAGCAG-3′ (SEQ ID NO: 4912)  5′-UCUUGGAUAUUCUCGACACAGCAgg-3′ (SEQ ID NO: 4670)3′-AGAGAACCUAUAAGAGCUGUGUCGUCC-5′ (SEQ ID NO: 4427) KRAS-334 Target:5′-TCTCTTGGATATTCTCGACACAGCAGG-3′ (SEQ ID NO: 4913)  5′-CUUGGAUAUUCUCGACACAGCAGgt-3′ (SEQ ID NO: 4671)3′-GAGAACCUAUAAGAGCUGUGUCGUCCA-5′ (SEQ ID NO: 4428) KRAS-335 Target:5′-CTCTTGGATATTCTCGACACAGCAGGT-3′ (SEQ ID NO: 4914)  5′-UUGGAUAUUCUCGACACAGCAGGtc-3′ (SEQ ID NO: 4672)3′-AGAACCUAUAAGAGCUGUGUCGUCCAG-5′ (SEQ ID NO: 4429) KRAS-336 Target:5′-TCTTGGATATTCTCGACACAGCAGGTC-3′ (SEQ ID NO: 4915)  5′-CAGCAGGUCAAGAGGAGUACAGUgc-3′ (SEQ ID NO: 4673)3′-GUGUCGUCCAGUUCUCCUCAUGUCACG-5′ (SEQ ID NO: 4430) KRAS-352 Target:5′-CACAGCAGGTCAAGAGGAGTACAGTGC-3′ (SEQ ID NO: 4916)  5′-AGCAGGUCAAGAGGAGUACAGUGca-3′ (SEQ ID NO: 4674)3′-UGUCGUCCAGUUCUCCUCAUGUCACGU-5′ (SEQ ID NO: 4431) KRAS-353 Target:5′-ACAGCAGGTCAAGAGGAGTACAGTGCA-3′ (SEQ ID NO: 4917)  5′-GCAGGUCAAGAGGAGUACAGUGCaa-3′ (SEQ ID NO: 4675)3′-GUCGUCCAGUUCUCCUCAUGUCACGUU-5′ (SEQ ID NO: 4432) KRAS-354 Target:5′-CAGCAGGTCAAGAGGAGTACAGTGCAA-3′ (SEQ ID NO: 4918)  5′-AGGAGUACAGUGCAAUGAGGGACca-3′ (SEQ ID NO: 4676)3′-UCUCCUCAUGUCACGUUACUCCCUGGU-5′ (SEQ ID NO: 4433) KRAS-364 Target:5′-AGAGGAGTACAGTGCAATGAGGGACCA-3′ (SEQ ID NO: 4919)  5′-GGAGUACAGUGCAAUGAGGGACCag-3′ (SEQ ID NO: 4677)3′-CUCCUCAUGUCACGUUACUCCCUGGUC-5′ (SEQ ID NO: 4434) KRAS-365 Target:5′-GAGGAGTACAGTGCAATGAGGGACCAG-3′ (SEQ ID NO: 4920)  5′-GAGUACAGUGCAAUGAGGGACCAgt-3′ (SEQ ID NO: 4678)3′-UCCUCAUGUCACGUUACUCCCUGGUCA-5′ (SEQ ID NO: 4435) KRAS-366 Target:5′-AGGAGTACAGTGCAATGAGGGACCAGT-3′ (SEQ ID NO: 4921)  5′-AGUACAGUGCAAUGAGGGACCAGta-3′ (SEQ ID NO: 4679)3′-CCUCAUGUCACGUUACUCCCUGGUCAU-5′ (SEQ ID NO: 4436) KRAS-367 Target:5′-GGAGTACAGTGCAATGAGGGACCAGTA-3′ (SEQ ID NO: 4922)  5′-GUACAGUGCAAUGAGGGACCAGUac-3′ (SEQ ID NO: 4680)3′-CUCAUGUCACGUUACUCCCUGGUCAUG-5′ (SEQ ID NO: 4437) KRAS-368 Target:5′-GAGTACAGTGCAATGAGGGACCAGTAC-3′ (SEQ ID NO: 4923)  5′-UACAGUGCAAUGAGGGACCAGUAca-3′ (SEQ ID NO: 4681)3′-UCAUGUCACGUUACUCCCUGGUCAUGU-5′ (SEQ ID NO: 4438) KRAS-369 Target:5′-AGTACAGTGCAATGAGGGACCAGTACA-3′ (SEQ ID NO: 4924)  5′-ACAGUGCAAUGAGGGACCAGUACat-3′ (SEQ ID NO: 4682)3′-CAUGUCACGUUACUCCCUGGUCAUGUA-5′ (SEQ ID NO: 4439) KRAS-370 Target:5′-GTACAGTGCAATGAGGGACCAGTACAT-3′ (SEQ ID NO: 4925)  5′-CAGUGCAAUGAGGGACCAGUACAtg-3′ (SEQ ID NO: 4683)3′-AUGUCACGUUACUCCCUGGUCAUGUAC-5′ (SEQ ID NO: 4440) KRAS-371 Target:5′-TACAGTGCAATGAGGGACCAGTACATG-3′ (SEQ ID NO: 4926)  5′-AGUGCAAUGAGGGACCAGUACAUga-3′ (SEQ ID NO: 4684)3′-UGUCACGUUACUCCCUGGUCAUGUACU-5′ (SEQ ID NO: 4441) KRAS-372 Target:5′-ACAGTGCAATGAGGGACCAGTACATGA-3′ (SEQ ID NO: 4927)  5′-GUAUUUGCCAUAAAUAAUACUAAat-3′ (SEQ ID NO: 4685)3′-CACAUAAACGGUAUUUAUUAUGAUUUA-5′ (SEQ ID NO: 4442) KRAS-420 Target:5′-GTGTATTTGCCATAAATAATACTAAAT-3′ (SEQ ID NO: 4928)  5′-UAUUUGCCAUAAAUAAUACUAAAtc-3′ (SEQ ID NO: 4686)3′-ACAUAAACGGUAUUUAUUAUGAUUUAG-5′ (SEQ ID NO: 4443) KRAS-421 Target:5′-TGTATTTGCCATAAATAATACTAAATC-3′ (SEQ ID NO: 4929)  5′-AUUUGCCAUAAAUAAUACUAAAUca-3′ (SEQ ID NO: 4687)3′-CAUAAACGGUAUUUAUUAUGAUUUAGU-5′ (SEQ ID NO: 4444) KRAS-422 Target:5′-GTATTTGCCATAAATAATACTAAATCA-3′ (SEQ ID NO: 4930)  5′-UUUGCCAUAAAUAAUACUAAAUCat-3′ (SEQ ID NO: 4688)3′-AUAAACGGUAUUUAUUAUGAUUUAGUA-5′ (SEQ ID NO: 4445) KRAS-423 Target:5′-TATTTGCCATAAATAATACTAAATCAT-3′ (SEQ ID NO: 4931)  5′-UUGCCAUAAAUAAUACUAAAUCAtt-3′ (SEQ ID NO: 4689)3′-UAAACGGUAUUUAUUAUGAUUUAGUAA-5′ (SEQ ID NO: 4446) KRAS-424 Target:5′-ATTTGCCATAAATAATACTAAATCATT-3′ (SEQ ID NO: 4932)  5′-UGCCAUAAAUAAUACUAAAUCAUtt-3′ (SEQ ID NO: 4690)3′-AAACGGUAUUUAUUAUGAUUUAGUAAA-5′ (SEQ ID NO: 4447) KRAS-425 Target:5′-TTTGCCATAAATAATACTAAATCATTT-3′ (SEQ ID NO: 4933)  5′-GCCAUAAAUAAUACUAAAUCAUUtg-3′ (SEQ ID NO: 4691)3′-AACGGUAUUUAUUAUGAUUUAGUAAAC-5′ (SEQ ID NO: 4448) KRAS-426 Target:5′-TTGCCATAAATAATACTAAATCATTTG-3′ (SEQ ID NO: 4934)  5′-AUACUAAAUCAUUUGAAGAUAUUca-3′ (SEQ ID NO: 4692)3′-AUUAUGAUUUAGUAAACUUCUAUAAGU-5′ (SEQ ID NO: 4449) KRAS-436 Target:5′-TAATACTAAATCATTTGAAGATATTCA-3′ (SEQ ID NO: 4935)  5′-UACUAAAUCAUUUGAAGAUAUUCac-3′ (SEQ ID NO: 4693)3′-UUAUGAUUUAGUAAACUUCUAUAAGUG-5′ (SEQ ID NO: 4450) KRAS-437 Target:5′-AATACTAAATCATTTGAAGATATTCAC-3′ (SEQ ID NO: 4936)  5′-ACUAAAUCAUUUGAAGAUAUUCAcc-3′ (SEQ ID NO: 4694)3′-UAUGAUUUAGUAAACUUCUAUAAGUGG-5′ (SEQ ID NO: 4451) KRAS-438 Target:5′-ATACTAAATCATTTGAAGATATTCACC-3′ (SEQ ID NO: 4937)  5′-CUAAAUCAUUUGAAGAUAUUCACca-3′ (SEQ ID NO: 4695)3′-AUGAUUUAGUAAACUUCUAUAAGUGGU-5′ (SEQ ID NO: 4452) KRAS-439 Target:5′-TACTAAATCATTTGAAGATATTCACCA-3′ (SEQ ID NO: 4938)  5′-UAAAUCAUUUGAAGAUAUUCACCat-3′ (SEQ ID NO: 4696)3′-UGAUUUAGUAAACUUCUAUAAGUGGUA-5′ (SEQ ID NO: 4453) KRAS-440 Target:5′-ACTAAATCATTTGAAGATATTCACCAT-3′ (SEQ ID NO: 4939)  5′-AAAUCAUUUGAAGAUAUUCACCAtt-3′ (SEQ ID NO: 4697)3′-GAUUUAGUAAACUUCUAUAAGUGGUAA-5′ (SEQ ID NO: 4454) KRAS-441 Target:5′-CTAAATCATTTGAAGATATTCACCATT-3′ (SEQ ID NO: 4940)  5′-AAUCAUUUGAAGAUAUUCACCAUta-3′ (SEQ ID NO: 4698)3′-AUUUAGUAAACUUCUAUAAGUGGUAAU-5′ (SEQ ID NO: 4455) KRAS-442 Target:5′-TAAATCATTTGAAGATATTCACCATTA-3′ {SEQ ID NO: 4941)  5′-AUCAUUUGAAGAUAUUCACCAUUat-3′ (SEQ ID NO: 4699)3′-UUUAGUAAACUUCUAUAAGUGGUAAUA-5′ (SEQ ID NO: 4456) KRAS-443 Target:5′-AAATCATTTGAAGATATTCACCATTAT-3′ (SEQ ID NO: 4942)  5′-UCAUUUGAAGAUAUUCACCAUUAta-3′ (SEQ ID NO: 4700)3′-UUAGUAAACUUCUAUAAGUGGUAAUAU-5′ (SEQ ID NO: 4457) KRAS-444 Target:5′-AATCATTTGAAGATATTCACCATTATA-3′ (SEQ ID NO: 4943)  5′-AUAUUCACCAUUAUAGAGAACAAat-3′ (SEQ ID NO: 4701)3′-UCUAUAAGUGGUAAUAUCUCUUGUUUA-5′ (SEQ ID NO: 4458) KRAS-454 Target:5′-AGATATTCACCATTATAGAGAACAAAT-3′ (SEQ ID NO: 4944)  5′-UAUUCACCAUUAUAGAGAACAAAtt-3′ (SEQ ID NO: 4702)3′-CUAUAAGUGGUAAUAUCUCUUGUUUAA-5′ (SEQ ID NO: 4459) KRAS-455 Target:5′-GATATTCACCATTATAGAGAACAAATT-3′ (SEQ ID NO: 4945)  5′-AUUCACCAUUAUAGAGAACAAAUta-3′ (SEQ ID NO: 4703)3′-UAUAAGUGGUAAUAUCUCUUGUUUAAU-5′ (SEQ ID NO: 4460) KRAS-456 Target:5′-ATATTCACCATTATAGAGAACAAATTA-3′ (SEQ ID NO: 4946)  5′-UUCACCAUUAUAGAGAACAAAUUaa-3′ (SEQ ID NO: 4704)3′-AUAAGUGGUAAUAUCUCUUGUUUAAUU-5′ (SEQ ID NO: 4461) KRAS-457 Target:5′-TATTCACCATTATAGAGAACAAATTAA-3′ (SEQ ID NO: 4947)  5′-UCACCAUUAUAGAGAACAAAUUAaa-3′ (SEQ ID NO: 4705)3′-UAAGUGGUAAUAUCUCUUGUUUAAUUU-5′ (SEQ ID NO: 4462) KRAS-458 Target:5′-ATTCACCATTATAGAGAACAAATTAAA-3′ (SEQ ID NO: 4948)  5′-CACCAUUAUAGAGAACAAAUUAAaa-3′ (SEQ ID NO: 4706)3′-AAGUGGUAAUAUCUCUUGUUUAAUUUU-5′ (SEQ ID NO: 4463) KRAS-459 Target:5′-TTCACCATTATAGAGAACAAATTAAAA-3′ (SEQ ID NO: 4949)  5′-ACCAUUAUAGAGAACAAAUUAAAag-3′ (SEQ ID NO: 4707)3′-AGUGGUAAUAUCUCUUGUUUAAUUUUC-5′ (SEQ ID NO: 4464) KRAS-460 Target:5′-TCACCATTATAGAGAACAAATTAAAAG-3′ (SEQ ID NO: 4950)  5′-CCAUUAUAGAGAACAAAUUAAAAga-3′ (SEQ ID NO: 4708)3′-GUGGUAAUAUCUCUUGUUUAAUUUUCU-5′ (SEQ ID NO: 4465) KRAS-461 Target:5′-CACCATTATAGAGAACAAATTAAAAGA-3′ (SEQ ID NO: 4951)  5′-CAUUAUAGAGAACAAAUUAAAAGag-3′ (SEQ ID NO: 4709)3′-UGGUAAUAUCUCUUGUUUAAUUUUCUC-5′ (SEQ ID NO: 4466) KRAS-462 Target:5′-ACCATTATAGAGAACAAATTAAAAGAG-3′ (SEQ ID NO: 4952)  5′-CUAUGGUCCUAGUAGGAAAUAAAtg-3′ (SEQ ID NO: 4710)3′-UGGAUACCAGGAUCAUCCUUUAUUUAC-5′ (SEQ ID NO: 4467) KRAS-508 Target:5′-ACCTATGGTCCTAGTAGGAAATAAATG-3′ (SEQ ID NO: 4953)  5′-UGUGAUUUGCCUUCUAGAACAGUag-3′ (SEQ ID NO: 4711)3′-UUACACUAAACGGAAGAUCUUGUCAUC-5′ (SEQ ID NO: 4468) KRAS-531 Target:5′-AATGTGATTTGCCTTCTAGAACAGTAG-3′ (SEQ ID NO: 4954)  5′-GUGAUUUGCCUUCUAGAACAGUAga-3′ (SEQ ID NO: 4712)3′-UACACUAAACGGAAGAUCUUGUCAUCU-5′ (SEQ ID NO: 4469) KRAS-532 Target:5′-ATGTGATTTGCCTTCTAGAACAGTAGA-3′ (SEQ ID NO: 4955)  5′-GAUUUGCCUUCUAGAACAGUAGAca-3′ (SEQ ID NO: 4713)3′-CACUAAACGGAAGAUCUUGUCAUCUGU-5′ (SEQ ID NO: 4470) KRAS-534 Target:5′-GTGATTTGCCTTCTAGAACAGTAGACA-3′ (SEQ ID NO: 4956)  5′-GUUAUGGAAUUCCUUUUAUUGAAac-3′ (SEQ ID NO: 4714)3′-UUCAAUACCUUAAGGAAAAUAACUUUG-5′ (SEQ ID NO: 4471) KRAS-586 Target:5′-AAGTTATGGAATTCCTTTTATTGAAAC-3′ (SEQ ID NO: 4957)  5′-UUAUGGAAUUCCUUUUAUUGAAAca-3′ (SEQ ID NO: 4715)3′-UCAAUACCUUAAGGAAAAUAACUUUGU-5′ (SEQ ID NO: 4472) KRAS-587 Target:5′-AGTTATGGAATTCCTTTTATTGAAACA-3′ (SEQ ID NO: 4958)  5′-UAUGGAAUUCCUUUUAUUGAAACat-3′ (SEQ ID NO: 4716)3′-CAAUACCUUAAGGAAAAUAACUUUGUA-5′ (SEQ ID NO: 4473) KRAS-588 Target:5′-GTTATGGAATTCCTTTTATTGAAACAT-3′ (SEQ ID NO: 4959)  5′-GAUGCCUUCUAUACAUUAGUUCGag-3′ (SEQ ID NO: 4717)3′-UACUACGGAAGAUAUGUAAUCAAGCUC-5′ (SEQ ID NO: 4474) KRAS-763 Target:5′-ATGATGCCTTCTATACATTAGTTCGAG-3′ (SEQ ID NO: 4960)  5′-AUGCCUUCUAUACAUUAGUUCGAga-3′ (SEQ ID NO: 4718)3′-ACUACGGAAGAUAUGUAAUCAAGCUCU-5′ (SEQ ID NO: 4475) KRAS-764 Target:5′-TGATGCCTTCTATACATTAGTTCGAGA-3′ (SEQ ID NO: 4961)  5′-CGAGAAAUUCGAAAACAUAAAGAaa-3′ (SEQ ID NO: 4719)3′-AAGCUCUUUAAGCUUUUGUAUUUCUUU-5′ (SEQ ID NO: 4476) KRAS-784 Target:5′-TTCGAGAAATTCGAAAACATAAAGAAA-3′ (SEQ ID NO: 4962)  5′-GAAAACAUAAAGAAAAGAUGAGCaa-3′ (SEQ ID NO: 4720)3′-AGCUUUUGUAUUUCUUUUCUACUCGUU-5′ (SEQ ID NO: 4477) KRAS-794 Target:5′-TCGAAAACATAAAGAAAAGATGAGCAA-3′ (SEQ ID NO: 4963)  5′-AAAACAUAAAGAAAAGAUGAGCAaa-3′ (SEQ ID NO: 4721)3′-GCUUUUGUAUUUCUUUUCUACUCGUUU-5′ (SEQ ID NO: 4478) KRAS-795 Target:5′-CGAAAACATAAAGAAAAGATGAGCAAA-3′ (SEQ ID NO: 4964)  5′-AAACAUAAAGAAAAGAUGAGCAAag-3′ (SEQ ID NO: 4722)3′-CUUUUGUAUUUCUUUUCUACUCGUUUC-5′ (SEQ ID NO: 4479) KRAS-796 Target:5′-GAAAACATAAAGAAAAGATGAGCAAAG-3′ (SEQ ID NO: 4965)  5′-AACAUAAAGAAAAGAUGAGCAAAga-3′ (SEQ ID NO: 4723)3′-UUUUGUAUUUCUUUUCUACUCGUUUCU-5′ (SEQ ID NO: 4480) KRAS-797 Target:5′-AAAACATAAAGAAAAGATGAGCAAAGA-3′ (SEQ ID NO: 4966)  5′-ACAUAAAGAAAAGAUGAGCAAAGat-3′ (SEQ ID NO: 4724)3′-UUUGUAUUUCUUUUCUACUCGUUUCUA-5′ (SEQ ID NO: 4481) KRAS-798 Target:5′-AAACATAAAGAAAAGATGAGCAAAGAT-3′ (SEQ ID NO: 4967)  5′-CAUAAAGAAAAGAUGAGCAAAGAtg-3′ (SEQ ID NO: 4725)3′-UUGUAUUUCUUUUCUACUCGUUUCUAC-5′ (SEQ ID.NO: 4482) KRAS-799 Target:5′-AACATAAAGAAAAGATGAGCAAAGATG-3′ (SEQ ID NO: 4968)  5′-AUAAAGAAAAGAUGAGCAAAGAUgg-3′ (SEQ ID NO: 4726)3′-UGUAUUUCUUUUCUACUCGUUUCUACC-5′ (SEQ ID NO: 4483) KRAS-800 Target:5′-ACATAAAGAAAAGATGAGCAAAGATGG-3′ (SEQ ID NO: 4969)  5′-UAAAGAAAAGAUGAGCAAAGAUGgt-3′ (SEQ ID NO: 4727)3′-GUAUUUCUUUUCUACUCGUUUCUACCA-5′ (SEQ ID NO: 4484) KRAS-801 Target:5′-CATAAAGAAAAGATGAGCAAAGATGGT-3′ (SEQ ID NO: 4970)  5′-AAAGAAAAGAUGAGCAAAGAUGGta-3′ (SEQ ID NO: 4728)3′-UAUUUCUUUUCUACUCGUUUCUACCAU-5′ (SEQ ID NO: 4485) KRAS-802 Target:5′-ATAAAGAAAAGATGAGCAAAGATGGTA-3′ (SEQ ID NO: 4971)  5′-AAGUGGUAAUUUUUGUACAUUACac-3′ (SEQ ID NO: 4729)3′-UGUUCACCAUUAAAAACAUGUAAUGUG-5′ (SEQ ID NO: 4486) KRAS-908 Target:5′-ACAAGTGGTAATTTTTGTACATTACAC-3′ (SEQ ID NO: 4972)  5′-AGUGGUAAUUUUUGUACAUUACAct-3′ (SEQ ID NO: 4730)3′-GUUCACCAUUAAAAACAUGUAAUGUGA-5′ (SEQ ID NO: 4487) KRAS-909 Target:5′-CAAGTGGTAATTTTTGTACATTACACT-3′ (SEQ ID NO: 4973)  5′-UUGUACAUUACACUAAAUUAUUAgc-3′ (SEQ ID NO: 4731)3′-AAAACAUGUAAUGUGAUUUAAUAAUCG-5′ (SEQ ID NO: 4488) KRAS-920 Target:5′-TTTTGTACATTACACTAAATTATTAGC-3′ (SEQ ID NO: 4974)  5′-UGUACAUUACACUAAAUUAUUAGca-3′ (SEQ ID NO: 4732)3′-AAACAUGUAAUGUGAUUUAAUAAUCGU-5′ (SEQ ID NO: 4489) KRAS-921 Target:5′-TTTGTACATTACACTAAATTATTAGCA-3′ (SEQ ID NO: 4975)  5′-GUACAUUACACUAAAUUAUUAGCat-3′ (SEQ ID NO: 4733)3′-AACAUGUAAUGUGAUUUAAUAAUCGUA-5′ (SEQ ID NO: 4490) KRAS-922 Target:5′-TTGTACATTACACTAAATTATTAGCAT-3′ (SEQ ID NO: 4976)  5′-UACAUUACACUAAAUUAUUAGCAtt-3′ (SEQ ID NO: 4734)3′-ACAUGUAAUGUGAUUUAAUAAUCGUAA-5′ (SEQ ID NO: 4491) KRAS-923 Target:5′-TGTACATTACACTAAATTATTAGCATT-3′ (SEQ ID NO: 4977)  5′-ACAUUACACUAAAUUAUUAGCAUtt-3′ (SEQ ID NO: 4735)3′-CAUGUAAUGUGAUUUAAUAAUCGUAAA-5′ (SEQ ID NO: 4492) KRAS-924 Target:5′-GTACATTACACTAAATTATTAGCATTT-3′ (SEQ ID NO: 4978)  5′-CAUUACACUAAAUUAUUAGCAUUtg-3′ (SEQ ID NO: 4736)3′-AUGUAAUGUGAUUUAAUAAUCGUAAAC-5′ (SEQ ID NO: 4493) KRAS-925 Target:5′-TACATTACACTAAATTATTAGCATTTG-3′ (SEQ ID NO: 4979)  5′-AUUACACUAAAUUAUUAGCAUUUgt-3′ (SEQ ID NO: 4737)3′-UGUAAUGUGAUU0AAUAAUCGUAAACA-5′ (SEQ ID NO: 4494) KRAS-926 Target:5′-ACATTACACTAAATTATTAGCATTTGT-3′ (SEQ ID NO: 4980)  5′-UUACACUAAAUUAUUAGCAUUUGtt-3′ (SEQ ID NO: 4738)3′-GUAAUGUGAUUUAAUAAUCGUAAACAA-5′ (SEQ ID NO: 4495) KRAS-927 Target:5′-CATTACACTAAATTATTAGCATTTGTT-3′ (SEQ ID NO: 4981)  5′-UACACUAAAUUAUUAGCAUUUGUtt-3′ (SEQ ID NO: 4739)3′-UAAUGUGAUUUAAUAAUCGUAAACAAA-5′ (SEQ ID NO: 4496) KRAS-928 Target:5′-ATTACACTAAATTATTAGCATTTGTTT-3′ (SEQ ID NO: 4982)  5′-UAUUAGCAUUUGUUUUAGCAUUAcc-3′ (SEQ ID NO: 4740)3′-UAAUAAUCGUAAACAAAAUCGUAAUGG-5′ (SEQ ID NO: 4497) KRAS-938 Target:5′-ATTATTAGCATTTGTTTTAGCATTACC-3′ (SEQ ID NO: 4983)  5′-AUUAGCAUUUGUUUUAGCAUUACct-3′ (SEQ ID NO: 4741)3′-AAUAAUCGUAAACAAAAUCGUAAUGGA-5′ (SEQ ID NO: 4498) KRAS-939 Target:5′-TTATTAGCATTTGTTTTAGCATTACCT-3′ (SEQ ID NO: 4984)  5′-UUAGCAUUUGUUUUAGCAUUACCta-3′ (SEQ ID NO: 4742)3′-AUAAUCGUAAACAAAAUCGUAAUGGAU-5′ (SEQ ID NO: 4499) KRAS-940 Target:5′-TATTAGCATTTGTTTTAGCATTACCTA-3′ (SEQ ID NO: 4985)  5′-UAGCAUUUGUUUUAGCAUUACCUaa-3′ (SEQ ID NO: 4743)3′-UAAUCGUAAACAAAAUCGUAAUGGAUU-5′ (SEQ ID NO: 4500) KRAS-941 Target:5′-ATTAGCATTTGTTTTAGCATTACCTAA-3′ (SEQ ID NO: 4986)  5′-AGCAUUUGUUUUAGCAUUACCUAat-3′ (SEQ ID NO: 4744)3′-AAUCGUAAACAAAAUCGUAAUGGAUUA-5′ (SEQ ID NO: 4501) KRAS-942 Target:5′-TTAGCATTTGTTTTAGCATTACCTAAT-3′ (SEQ ID NO: 4987)  5′-GCAUUUGUUUUAGCAUUACCUAAtt-3′ (SEQ ID NO: 4745)3′-AUCGUAAACAAAAUCGUAAUGGAUUAA-5′ (SEQ ID NO: 4502) KRAS-943 Target:5′-TAGCATTTGTTTTAGCATTACCTAATT-3′ (SEQ ID NO: 4988)  5′-CAUUUGUUUUAGCAUUACCUAAUtt-3′ (SEQ ID NO: 4746)3′-UCGUAAACAAAAUCGUAAUGGAUUAAA-5′ (SEQ ID NO: 4503) KRAS-944 Target:5′-AGCATTTGTTTTAGCATTACCTAATTT-3′ (SEQ ID NO: 4989)  5′-AUUUGUUUUAGCAUUACCUAAUUtt-3′ (SEQ ID NO: 4747)3′-CGUAAACAAAAUCGUAAUGGAUUAAAA-5′ (SEQ ID NO: 4504) KRAS-945 Target:5′-GCATTTGTTTTAGCATTACCTAATTTT-3′ (SEQ ID NO: 4990)  5′-UUUGUUUUAGCAUUACCUAAUUUtt-3′ (SEQ ID NO: 4748)3′-GUAAACAAAAUCGUAAUGGAUUAAAAA-5′ (SEQ ID NO: 4505) KRAS-946 Target:5′-CATTTGTTTTAGCATTACCTAATTTTT-3′ (SEQ ID NO: 4991)  5′-CUUAUUUUAAAAUGACAGUGGAAgt-3′ (SEQ ID NO: 4749)3′-ACGAAUAAAAUUUUACUGUCACCUUCA-5′ (SEQ ID NO: 4506) KRAS-1010 Target:5′-TGCTTATTTTAAAATGACAGTGGAAGT-3′ (SEQ ID NO: 4992)  5′-UAUUUUAAAAUGACAGUGGAAGUtt-3′ (SEQ ID NO: 4750)3′-GAAUAAAAUUUUACUGUCACCUUCAAA-5′ (SEQ ID NO: 4507) KRAS-1012 Target:5′-CTTATTTTAAAATGACAGTGGAAGTTT-3′ (SEQ ID NO: 4993)  5′-UCUAAGUGCCAGUAUUCCCAGAGtt-3′ (SEQ ID NO: 4751)3′-GGAGAUUCACGGUCAUAAGGGUCUCAA-5′ (SEQ ID NO: 4508) KRAS-1045 Target:5′-CCTCTAAGTGCCAGTATTCCCAGAGTT-3′ (SEQ ID NO: 4994)  5′-CAAAUUAAUGAAGCUUUUGAAUCat-3′ (SEQ ID NO: 4752)3′-UUGUUUAAUUACUUCGAAAACUUAGUA-5′ (SEQ ID NO: 4509) KRAS-1197 Target:5′-AACAAATTAATGAAGCTTTTGAATCAT-3′ (SEQ ID NO: 4995)  5′-AAAUUAAUGAAGCUUUUGAAUCAtc-3′ (SEQ ID NO: 4753)3′-UGUUUAAUUACUUCGAAAACUUAGUAG-5′ (SEQ ID NO: 4510) KRAS-1198 Target:5′-ACAAATTAATGAAGCTTTTGAATCATC-3′ (SEQ ID NO: 4996)  5′-UGUGUUUUAUCUAGUCACAUAAAtg-3′ (SEQ ID NO: 4754)3′-AGACACAAAAUAGAUCAGUGUAUUUAC-5′ (SEQ ID NO: 4511) KRAS-1230 Target:5′-TCTGTGTTTTATCTAGTCACATAAATG-3′ (SEQ ID NO: 4997)  5′-GUGUUUUAUCUAGUCACAUAAAUgg-3′ (SEQ ID NO: 4755)3′-GACACAAAAUAGAUCAGUGUAUUUACC-5′ (SEQ ID NO: 4512) KRAS-1231 Target:5′-CTGTGTTTTATCTAGTCACATAAATGG-3′ (SEQ ID NO: 4998)  5′-UUUUAUCUAGUCACAUAAAUGGAtt-3′ (SEQ ID NO: 4756)3′-ACAAAAUAGAUCAGUGUAUUUACCUAA-5′ (SEQ ID NO: 4513) KRAS-1234 Target:5′-TGTTTTATCTAGTCACATAAATGGATT-3′ (SEQ ID NO: 4999)  5′-UAAAUGGAUUAAUUACUAAUUUCag-3′ (SEQ ID NO: 4757)3′-GUAUUUACCUAAUUAAUGAUUAAAGUC-5′ (SEQ ID NO: 4514) KRAS-1249 Target:5′-CATAAATGGATTAATTACTAATTTCAG-3′ (SEQ ID NO: 5000)  5′-AAAUGGAUUAAUUACUAAUUUCAgt-3′ (SEQ ID NO: 4758)3′-UAUUUACCUAAUUAAUGAUUAAAGUCA-5′ (SEQ ID NO: 4515) KRAS-1250 Target:5′-ATAAATGGATTAATTACTAATTTCAGT-3′ (SEQ ID NO: 5001)  5′-AUUGGUUUUUACUGAAACAUUGAgg-3′ (SEQ ID NO: 4759)3′-AUUAACCAAAAAUGACUUUGUAACUCC-5′ (SEQ ID NO: 4516) KRAS-1287 Target:5′-TAATTGGTTTTTACTGAAACATTGAGG-3′ (SEQ ID NO: 5002)  5′-CAUUUCCUUUUCACAUUAGAUAAat-3′ (SEQ ID NO: 4760)3′-CGGUAAAGGAAAAGUGUAAUCUAUUUA-5′ (SEQ ID NO: 4517) KRAS-1527 Target:5′-GCCATTTCCTTTTCACATTAGATAAAT-3′ (SEQ ID NO: 5003)  5′-CUUUUCACAUUAGAUAAAUUACUat-3′ (SEQ ID NO: 4761)3′-AGGAAAAGUGUAAUCUAUUUAAUGAUA-5′ (SEQ ID NO: 4518) KRAS-1533 Target:5′-TCCTTTTCACATTAGATAAATTACTAT-3′ (SEQ ID NO: 5004)  5′-CAUUAGAUAAAUUACUAUAAAGAct-3′ (SEQ ID NO: 4762)3′-GUGUAAUCUAUUUAAUGAUAUUUCUGA-5′ (SEQ ID NO: 4519) KRAS-1540 Target:5′-CACATTAGATAAATTACTATAAAGACT-3′ (SEQ ID NO: 5005)  5′-AUUAGAUAAAUUACUAUAAAGACtc-3′ (SEQ ID NO: 4763)3′-UGUAAUCUAUUUAAUGAUAUUUCUGAG-5′ (SEQ ID NO: 4520) KRAS-1541 Target:5′-ACATTAGATAAATTACTATAAAGACTC-3′ (SEQ ID NO: 5006)  5′-UUAGAUAAAUUACUAUAAAGACUcc-3′ (SEQ ID NO: 4764)3′-GUAAUCUAUUUAAUGAUAUUUCUGAGG-5′ (SEQ ID NO: 4521) KRAS-1542 Target:5′-CATTAGATAAATTACTATAAAGACTCC-3′ (SEQ ID NO: 5007)  5′-UAAGGCAGACCCAGUAUGAAAUGgg-3′ (SEQ ID NO: 4765)3′-CAAUUCCGUCUGGGUCAUACUUUACCC-5′ (SEQ ID NO: 4522) KRAS-1583 Target:5′-GTTAAGGCAGACCCAGTATGAAATGGG-3′ (SEQ ID NO: 5008)  5′-AAGGCAGACCCAGUAUGAAAUGGgg-3′ (SEQ ID NO: 4766)3′-AAUUCCGUCUGGGUCAUACUUUACCCC-5′ (SEQ ID NO: 4523) KRAS-1584 Target:5′-TTAAGGCAGACCCAGTATGAAATGGGG-3′ (SEQ ID NO: 5009)  5′-AGGCAGACCCAGUAUGAAAUGGGga-3′ (SEQ ID NO: 4767)3′-AUUCCGUCUGGGUCAUACUUUACCCCU-5′ (SEQ ID NO: 4524) KRAS-1585 Target:5′-TAAGGCAGACCCAGTATGAAATGGGGA-3′ (SEQ ID NO: 5010)  5′-GGCAGACCCAGUAUGAAAUGGGGat-3′ (SEQ ID NO: 4768)3′-UUCCGUCUGGGUCAUACUUUACCCCUA-5′ (SEQ ID NO: 4525) KRAS-1586 Target:5′-AAGGCAGACCCAGTATGAAATGGGGAT-3′ (SEQ ID NO: 5011)  5′-UAUGAAAUGGGGAUUAUUAUAGCaa-3′ (SEQ ID NO: 4769)3′-UCAUACUUUACCCCUAAUAAUAUCGUU-5′ (SEQ ID NO: 4526) KRAS-1597 Target:5′-AGTATGAAATGGGGATTATTATAGCAA-3′ (SEQ ID NO: 5012)  5′-GGGAUUAUUAUAGCAACCAUUUUgg-3′ (SEQ ID NO: 4770)3′-ACCCCUAAUAAUAUCGUUGGUAAAACC-5′ (SEQ ID NO: 4527) KRAS-1606 Target:5′-TGGGGATTATTATAGCAACCATTTTGG-3′ (SEQ ID NO: 5013)  5′-GGGCUAUAUUUACAUGCUACUAAat-3′ (SEQ ID NO: 4771)3′-ACCCCGAUAUAAAUGUACGAUGAUUUA-5′ (SEQ ID NO: 4528) KRAS-1630 Target:5′-TGGGGCTATATTTACATGCTACTAAAT-3′ (SEQ ID NO: 5014)  5′-GGCUAUAUUUACAUGCUACUAAAtt-3′ (SEQ ID NO: 4772)3′-CCCCGAUAUAAAUGUACGAUGAUUUAA-5′ (SEQ ID NO: 4529) KRAS-1631 Target:5′-GGGGCTATATTTACATGCTACTAAATT-3′ (SEQ ID NO: 5015)  5′-GCUAUAUUUACAUGCUACUAAAUtt-3′ (SEQ ID NO: 4773)3′-CCCGAUAUAAAUGUACGAUGAUUUAAA-5′ (SEQ ID NO: 4530) KRAS-1632 Target:5′-GGGCTATATTTACATGCTACTAAATTT-3′ (SEQ ID NO: 5016)  5′-CUAUAUUUACAUGCUACUAAAUUtt-3′ (SEQ ID NO: 4774)3′-CCGAUAUAAAUGUACGAUGAUUUAAAA-5′ (SEQ ID NO: 4531) KRAS-1633 Target:5′-GGCTATATTTACATGCTACTAAATTTT-3′ (SEQ ID NO: 5017)  5′-UAUAUUUACAUGCUACUAAAUUUtt-3′ (SEQ ID NO: 4775)3′-CGAUAUAAAUGUACGAUGAUUUAAAAA-5′ (SEQ ID NO: 4532) KRAS-1634 Target:5′-GCTATATTTACATGCTACTAAATTTTT-3′ (SEQ ID NO: 5018)  5′-AUAUUUACAUGCUACUAAAUUUUta-3′ (SEQ ID NO: 4776)3′-GAUAUAAAUGUACGAUGAUUUAAAAAU-5′ (SEQ ID NO: 4533) KRAS-1635 Target:5′-CTATATTTACATGCTACTAAATTTTTA-3′ (SEQ ID NO: 5019)  5′-UAUUUACAUGCUACUAAAUUUUUat-3′ (SEQ ID NO: 4777)3′-AUAUAAAUGUACGAUGAUUUAAAAAUA-5′ (SEQ ID NO: 4534) KRAS-1636 Target:5′-TATATTTACATGCTACTAAATTTTTAT-3′ (SEQ ID NO: 5020)  5′-AUUUACAUGCUACUAAAUUUUUAta-3′ (SEQ ID NO: 4778)3′-UAUAAAUGUACGAUGAUUUAAAAAUAU-5′ (SEQ ID NO: 4535) KRAS-1637 Target:5′-ATATTTACATGCTACTAAATTTTTATA-3′ (SEQ ID NO: 5021)  5′-UUUACAUGCUACUAAAUUUUUAUaa-3′ (SEQ ID NO: 4779)3′-AUAAAUGUACGAUGAUUUAAAAAUAUU-5′ (SEQ ID NO: 4536) KRAS-1638 Target:5′-TATTTACATGCTACTAAATTTTTATAA-3′ (SEQ ID NO: 5022)  5′-UUACAUGCUACUAAAUUUUUAUAat-3′ (SEQ ID NO: 4780)3′-UAAAUGUACGAUGAUUUAAAAAUAUUA-5′ (SEQ ID NO: 4537) KRAS-1639 Target:5′-ATTTACATGCTACTAAATTTTTATAAT-3′ (SEQ ID NO: 5023)  5′-UACAUGCUACUAAAUUUUUAUAAta-3′ (SEQ ID NO: 4781)3′-AAAUGUACGAUGAUUUAAAAAUAUUAU-5′ (SEQ ID NO: 4538) KRAS-1640 Target:5′-TTTACATGCTACTAAATTTTTATAATA-3′ (SEQ ID NO: 5024)  5′-CUUUCAUAGUAUAACUUUAAAUCtt-3′ (SEQ ID NO: 4782)3′-GAGAAAGUAUCAUAUUGAAAUUUAGAA-5′ (SEQ ID NO: 4539) KRAS-1736 Target:5′-CTCTTTCATAGTATAACTTTAAATCTT-3′ (SEQ ID NO: 5025)  5′-AUAGUAUAACUUUAAAUCUUUUCtt-3′ (SEQ ID NO: 4783)3′-AGUAUCAUAUUGAAAUUUAGAAAAGAA-5′ (SEQ ID NO: 4540) KRAS-1741 Target:5′-TCATAGTATAACTTTAAATCTTTTCTT-3′ (SEQ ID NO: 5026)  5′-UAGUAUAACUUUAAAUCUUUUCUtc-3′ (SEQ ID NO: 4784)3′-GUAUCAUAUUGAAAUUUAGAAAAGAAG-5′ (SEQ ID NO: 4541) KRAS-1742 Target:5′-CATAGTATAACTTTAAATCTTTTCTTC-3′ (SEQ ID NO: 5027)  5′-UAAAUCUUUUCUUCAACUUGAGUct-3′ (SEQ ID NO: 4785)3′-AAAUUUAGAAAAGAAGUUGAACUCAGA-5′ (SEQ ID NO: 4542) KRAS-1753 Target:5′-TTTAAATCTTTTCTTCAACTTGAGTCT-3′ (SEQ ID NO: 5028)  5′-AAAUCUUUUCUUCAACUUGAGUCtt-3′ (SEQ ID NO: 4786)3′-AAUUUAGAAAAGAAGUUGAACUCAGAA-5′ (SEQ ID NO: 4543) KRAS-1754 Target:5′-TTAAATCTTTTCTTCAACTTGAGTCTT-3′ (SEQ ID NO: 5029)  5′-CUUGAGUCUUUGAAGAUAGUUUUaa-3′ (SEQ ID NO: 4787)3′-UUGAACUCAGAAACUUCUAUCAAAAUU-5′ (SEQ ID NO: 4544) KRAS-1769 Target:5′-AACTTGAGTCTTTGAAGATAGTTTTAA-3′ (SEQ ID NO: 5030)  5′-UGAGUCUUUGAAGAUAGUUUUAAtt-3′ (SEQ ID NO: 4788)3′-GAACUCAGAAACUUCUAUCAAAAUUAA-5′ (SEQ ID NO: 4545) KRAS-1771 Target:5′-CTTGAGTCTTTGAAGATAGTTTTAATT-3′ (SEQ ID NO: 5031)  5′-GAGUCUUUGAAGAUAGUUUUAAUtc-3′ (SEQ ID NO: 4789)3′-AACUCAGAAACUUCUAUCAAAAUUAAG-5′ (SEQ ID NO: 4546) KRAS-1772 Target:5′-TTGAGTCTTTGAAGATAGTTTTAATTC-3′ (SEQ ID NO: 5032)  5′-GAUAGUUUUAAUUCUGCUUGUGAca-3′ (SEQ ID NO: 4790)3′-UUCUAUCAAAAUUAAGACGAACACUGU-5′ (SEQ ID NO: 4547) KRAS-1783 Target:5′-AAGATAGTTTTAATTCTGCTTGTGACA-3′ (SEQ ID NO: 5033)  5′-AUAGUUUUAAUUCUGCUUGUGACat-3′ (SEQ ID NO: 4791)3′-UCUAUCAAAAUUAAGACGAACACUGUA-5′ (SEQ ID NO: 4548) KRAS-1784 Target:5′-AGATAGTTTTAATTCTGCTTGTGACAT-3′ (SEQ ID NO: 5034)  5′-UAGUUUUAAUUCUGCUUGUGACAtt-3′ (SEQ ID NO: 4792)3′-CUAUCAAAAUUAAGACGAACACUGUAA-5′ (SEQ ID NO: 4549) KRAS-1785 Target:5′-GATAGTTTTAATTCTGCTTGTGACATT-3′ (SEQ ID NO: 5035)  5′-CUUGUGACAUUAAAAGAUUAUUUgg-3′ (SEQ ID NO: 4793)3′-ACGAACACUGUAAUUUUCUAAUAAACC-5′ (SEQ ID NO: 4550) KRAS-1799 Target:5′-TGCTTGTGACATTAAAAGATTATTTGG-3′ (SEQ ID NO: 5036)  5′-UAUUAACUCAAAAGUUGAGAUUUtg-3′ (SEQ ID NO: 4794)3′-UUAUAAUUGAGUUUUCAACUCUAAAAC-5′ (SEQ ID NO: 4551) KRAS-2100 Target:5′-AATATTAACTCAAAAGTTGAGATTTTG-3′ (SEQ ID NO: 5037)  5′-UGUGCCAAGACAUUAAUUUUUUUtt-3′ (SEQ ID NO: 4795)3′-CCACACGGUUCUGUAAUUAAAAAAAAA-5′ (SEQ ID NO: 4552) KRAS-2134 Target:5′-GGTGTGCCAAGACATTAATTTTTTTTT-3′ (SEQ ID NO: 5038)  5′-UGGUUAAAUUAACAUUGCAUAAAca-3′ (SEQ ID NO: 4796)3′-UGACCAAUUUAAUUGUAACGUAUUUGU-5′ (SEQ ID NO: 4553) KRAS-2216 Target:5′-ACTGGTTAAATTAACATTGCATAAACA-3′ (SEQ ID NO: 5039)  5′-GGUUAAAUUAACAUUGCAUAAACac-3′ (SEQ ID NO: 4797)3′-GACCAAUUUAAUUGUAACGUAUUUGUG-5′ (SEQ ID NO: 4554) KRAS-2217 Target:5′-CTGGTTAAATTAACATTGCATAAACAC-3′ (SEQ ID NO: 5040)  5′-GUUAAAUUAACAUUGCAUAAACAct-3′ (SEQ ID NO:.4798)3′-ACCAAUUUAAUUGUAACGUAUUUGUGA-5′ (SEQ ID NO: 4555) KRAS-2218 Target:5′-TGGTTAAATTAACATTGCATAAACACT-3′ (SEQ ID NO: 5041)  5′-AUUGCAUAAACACUUUUCAAGUCtg-3′ (SEQ ID NO: 4799)3′-UGUAACGUAUUUGUGAAAAGUUCAGAC-5′ (SEQ ID NO: 4556) KRAS-2229 Target:5′-ACATTGCATAAACACTTTTCAAGTCTG-3′ (SEQ ID NO: 5042)  5′-AAGUCUGAUCCAUAUUUAAUAAUgc-3′ (SEQ ID NO: 4800)3′-AGUUCAGACUAGGUAUAAAUUAUUACG-5′ (SEQ ID NO: 4557) KRAS-2247 Target:5′-TCAAGTCTGATCCATATTTAATAATGC-3′ (SEQ ID NO: 5043)  5′-UAAAAUAAAUGAAGUGAGAUGGCat-3′ (SEQ ID NO: 4801)3′-AAAUUUUAUUUACUUCACUCUACCGUA-5′ (SEQ ID NO: 4558) KRAS-2326 Target:5′-TTTAAAATAAATGAAGTGAGATGGCAT-3′ (SEQ ID NO: 5044)  5′-AAAAUAAAUGAAGUGAGAUGGCAtg-3′ (SEQ ID NO: 4802)3′-AAUUUUAUUUACUUCACUCUACCGUAC-5′ (SEQ ID NO: 4559) KRAS-2327 Target:5′-TTAAAATAAATGAAGTGAGATGGCATG-3′ (SEQ ID NO: 5045)  5′-AAGCUCAGCACAAUCUGUAAAUUtt-3′ (SEQ ID NO: 4803)3′-AGUUCGAGUCGUGUUAGACAUUUAAAA-5′ (SEQ ID NO: 4560) KRAS-2547 Target:5′-TCAAGCTCAGCACAATCTGTAAATTTT-3′ (SEQ ID NO: 5046)  5′-AGCUCAGCACAAUCUGUAAAUUUtt-3′ (SEQ ID NO: 4804)3′-GUUCGAGUCGUGUUAGACAUUUAAAAA-5′ (SEQ ID NO: 4561) KRAS-2548 Target:5′-CAAGCTCAGCACAATCTGTAAATTTTT-3′ (SEQ ID NO: 5047)  5′-AUAACUGUGAUUCUUUUAGGACAat-3′ (SEQ ID NO: 4805)3′-CGUAUUGACACUAAGAAAAUCCUGUUA-5′ (SEQ ID NO: 4562) KRAS-3741 Target:5′-GCATAACTGTGATTCTTTTAGGACAAT-3′ (SEQ ID NO: 5048)  5′-UGUGAUUCUUUUAGGACAAUUACtg-3′ (SEQ ID NO: 4806)3′-UGACACUAAGAAAAUCCUGUUAAUGAC-5′ (SEQ ID NO: 4563) KRAS-3746 Target:5′-ACTGTGATTCTTTTAGGACAATTACTG-3′ (SEQ ID NO: 5049)  5′-GUGAUUCUUUUAGGACAAUUACUgt-3′ (SEQ ID NO: 4807)3′-GACACUAAGAAAAUCCUGUUAAUGACA-5′ (SEQ ID NO: 4564) KRAS-3747 Target:5′-CTGTGATTCTTTTAGGACAATTACTGT-3′ (SEQ ID NO: 5050)  5′-UGUAUGUCAGAUAUUCAUAUUGAcc-3′ (SEQ ID NO: 4808)3′-CCACAUACAGUCUAUAAGUAUAACUGG-5′ (SEQ ID NO: 4565) KRAS-3783 Target:5′-GGTGTATGTCAGATATTCATATTGACC-3′ (SEQ ID NO: 5051)  5′-GUAUGUCAGAUAUUCAUAUUGACcc-3′ (SEQ ID NO: 4809)3′-CACAUACAGUCUAUAAGUAUAACUGGG-5′ (SEQ ID NO: 4566) KRAS-3784 Target:5′-GTGTATGTCAGATATTCATATTGACCC-3′ (SEQ ID NO: 5052)  5′-AAUGUGUAAUAUUCCAGUUUUCUct-3′ (SEQ ID NO: 4810)3′-GUUUACACAUUAUAAGGUCAAAAGAGA-5′ (SEQ ID NO: 4567) KRAS-3810 Target:5′-CAAATGTGTAATATTCCAGTTTTCTCT-3′ (SEQ ID NO: 5053)  5′-CACUGCAUAGGAAUUUAGAACCUaa-3′ (SEQ ID NO: 4811)3′-GUGUGACGUAUCCUUAAAUCUUGGAUU-5′ (SEQ ID NO: 4568) KRAS-4396 Target:5′-CACACTGCATAGGAATTTAGAACCTAA-3′ (SEQ ID NO: 5054)  5′-CACCAUUGCACAAUUUUGUCCUAat-3′ (SEQ ID NO: 4812)3′-CAGUGGUAACGUGUUAAAACAGGAUUA-5′ (SEQ ID NO: 4569) KRAS-4447 Target:5′-GTCACCATTGCACAATTTTGTCCTAAT-3′ (SEQ ID NO: 5055)  5′-ACCAUUGCACAAUUUUGUCCUAAta-3′ (SEQ ID NO: 4813)3′-AGUGGUAACGUGUUAAAACAGGAUUAU-5′ (SEQ ID NO: 4570) KRAS-4448 Target:5′-TCACCATTGCACAATTTTGTCCTAATA-3′ (SEQ ID NO: 5056)  5′-CCAUUGCACAAUUUUGUCCUAAUat-3′ (SEQ ID NO: 4814)3′-GUGGUAACGUGUUAAAACAGGAUUAUA-5′ (SEQ ID NO: 4571) KRAS-4449 Target:5′-CACCATTGCACAATTTTGTCCTAATAT-3′ (SEQ ID NO: 5057)  5′-CAUUGCACAAUUUUGUCCUAAUAta-3′ (SEQ ID NO: 4815)3′-UGGUAACGUGUUAAAACAGGAUUAUAU-5′ (SEQ ID NO: 4572) KRAS-4450 Target:5′-ACCATTGCACAATTTTGTCCTAATATA-3′ (SEQ ID NO: 5058)  5′-AUUGCACAAUUUUGUCCUAAUAUat-3′ (SEQ ID NO: 4816)3′-GGUAACGUGUUAAAACAGGAUUAUAUA-5′ (SEQ ID NO: 4573) KRAS-4451 Target:5′-CCATTGCACAATTTTGTCCTAATATAT-3′ (SEQ ID NO: 5059)  5′-UUGCACAAUUUUGUCCUAAUAUAta-3′ (SEQ ID NO: 4817)3′-GUAACGUGUUAAAACAGGAUUAUAUAU-5′ (SEQ ID NO: 4574) KRAS-4452 Target:5′-CATTGCACAATTTTGTCCTAATATATA-3′ (SEQ ID NO: 5060)  5′-UAGCAUGAAUUCUGCAUUGAGAAac-3′ (SEQ ID NO: 4818)3′-CUAUCGUACUUAAGACGUAACUCUUUG-5′ (SEQ ID NO: 4575) KRAS-4748 Target:5′-GATAGCATGAATTCTGCATTGAGAAAC-3′ (SEQ ID NO: 5061)  5′-AGCAUGAAUUCUGCAUUGAGAAAct-3′ (SEQ ID NO: 4819)3′-UAUCGUACUUAAGACGUAACUCUUUGA-5′ (SEQ ID NO: 4576) KRAS-4749 Target:5′-ATAGCATGAATTCTGCATTGAGAAACT-3′ (SEQ ID NO: 5062)  5′-UUUGAAGUGCCUGUUUGGGAUAAtg-3′ (SEQ ID NO: 4820)3′-UCAAACUUCACGGACAAACCCUAUUAC-5′ (SEQ ID NO: 4577) KRAS-4878 Target:5′-AGTTTGAAGTGCCTGTTTGGGATAATG-3′ (SEQ ID NO: 5063)  5′-UUGAAGUGCCUGUUUGGGAUAAUga-3′ (SEQ ID NO: 4821)3′-CAAACUUCACGGACAAACCCUAUUACU-5′ (SEQ ID NO: 4578) KRAS-4879 Target:5′-GTTTGAAGTGCCTGTTTGGGATAATGA-3′ (SEQ ID NO: 5064)  5′-UGAAGUGCCUGUUUGGGAUAAUGat-3′ (SEQ ID NO: 4822)3′-AAACUUCACGGACAAACCCUAUUACUA-5′ (SEQ ID NO: 4579) KRAS-4880 Target:5′-TTTGAAGTGCCTGTTTGGGATAATGAT-3′ (SEQ ID NO: 5065)  5′-UUUGAGUGCCAAUUUCUUACUAGta-3′ (SEQ ID NO: 4823)3′-GGAAACUCACGGUUAAAGAAUGAUCAU-5′ (SEQ ID NO: 4580) KRAS-5073 Target:5′-CCTTTGAGTGCCAATTTCTTACTAGTA-3′ (SEQ ID NO: 5066)  5′-UUGAGUGCCAAUUUCUUACUAGUac-3′ (SEQ ID NO: 4824)3′-GAAACUCACGGUUAAAGAAUGAUCAUG-5′ (SEQ ID NO: 4581) KRAS-5074 Target:5′-CTTTGAGTGCCAATTTCTTACTAGTAC-3′ (SEQ ID NO: 5067)  5′-UGAGUGCCAAUUUCUUACUAGUAct-3′ (SEQ ID NO: 4825)3′-AAACUCACGGUUAAAGAAUGAUCAUGA-5′ (SEQ ID NO: 4582) KRAS-5075 Target:5′-TTTGAGTGCCAATTTCTTACTAGTACT-3′ (SEQ ID NO: 5068)  5′-GAGUGCCAAUUUCUUACUAGUACta-3′ (SEQ ID NO: 4826)3′-AACUCACGGUUAAAGAAUGAUCAUGAU-5′ (SEQ ID NO: 4583) KRAS-5076 Target:5′-TTGAGTGCCAATTTCTTACTAGTACTA-3′ (SEQ ID NO: 5069)  5′-AGUGCCAAUUUCUUACUAGUACUat-3′ (SEQ ID NO: 4827)3′-ACUCACGGUUAAAGAAUGAUCAUGAUA-5′ (SEQ ID NO: 4584) KRAS-5077 Target:5′-TGAGTGCCAATTTCTTACTAGTACTAT-3′ (SEQ ID NO: 5070)  5′-GUGCCAAUUUCUUACUAGUACUAtt-3′ (SEQ ID NO: 4828)3′-CUCACGGUUAAAGAAUGAUCAUGAUAA-5′ (SEQ ID NO: 4585) KRAS-5078 Target:5′-GAGTGCCAATTTCTTACTAGTACTATT-3′ (SEQ ID NO: 5071)  5′-AUGUAUUUUAACUAUUUUUGUAUag-3′ (SEQ ID NO: 4829)3′-CUUACAUAAAAUUGAUAAAAACAUAUC-5′ (SEQ ID NO: 4586) KRAS-5128 Target:5′-GAATGTATTTTAACTATTTTTGTATAG-3′ (SEQ ID NO: 5072)  5′-UGUAUUUUAACUAUUUUUGUAUAgt-3′ (SEQ ID NO: 4830)3′-UUACAUAAAAUUGAUAAAAACAUAUCA-5′ (SEQ ID NO: 4587) KRAS-5129 Target:5′-AATGTATTTTAACTATTTTTGTATAGT-3′ (SEQ ID NO: 5073)  5′-ACUAUUUUUGUAUAGUGUAAACUga-3′ (SEQ ID NO: 4831)3′-AUUGAUAAAAACAUAUCACAUUUGACU-5′ (SEQ ID NO: 4588) KRAS-5138 Target:5′-TAACTATTTTTGTATAGTGTAAACTGA-3′ (SEQ ID NO: 5074)  5′-CUAUUUUUGUAUAGUGUAAACUGaa-3′ (SEQ ID NO: 4832)3′-UUGAUAAAAACAUAUCACAUUUGACUU-5′ (SEQ ID NO: 4589) KRAS-5139 Target:5′-AACTATTTTTGTATAGTGTAAACTGAA-3′ (SEQ ID NO: 5075)  5′-UAUUUUUGUAUAGUGUAAACUGAaa-3′ (SEQ ID NO: 4833)3′-UGAUAAAAACAUAUCACAUUUGACUUU-5′ (SEQ ID NO: 4590) KRAS-5140 Target:5′-ACTATTTTTGTATAGTGTAAACTGAAA-3′ (SEQ ID NO: 5076)  5′-AUUUUUGUAUAGUGUAAACUGAAac-3′ (SEQ ID NO: 4834)3′-GAUAAAAACAUAUCACAUUUGACUUUG-5′ (SEQ ID NO: 4591) KRAS-5141 Target:5′-CTATTTTTGTATAGTGTAAACTGAAAC-3′ (SEQ ID NO: 5077)  5′-UUUUUGUAUAGUGUAAACUGAAAca-3′ (SEQ ID NO: 4835)3′-AUAAAAACAUAUCACAUUUGACUUUGU-5′ (SEQ ID NO: 4592) KRAS-5142 Target:5′-TATTTTTGTATAGTGTAAACTGAAACA-3′ (SEQ ID NO: 5078)  5′-UUUUGUAUAGUGUAAACUGAAACat-3′ (SEQ ID NO: 4836)3′-UAAAAACAUAUCACAUUUGACUUUGUA-5′ (SEQ ID NO: 4593) KRAS-5143 Target:5′-ATTTTTGTATAGTGTAAACTGAAACAT-3′ (SEQ ID NO: 5079)  5′-AACAUGCACAUUUUGUACAUUGUgc-3′ (SEQ ID NO: 4837)3′-CUUUGUACGUGUAAAACAUGUAACACG-5′ (SEQ ID NO: 4594) KRAS-5163 Target:5′-GAAACATGCACATTTTGTACATTGTGC-3′ (SEQ ID NO: 5080)  5′-ACAUGCACAUUUUGUACAUUGUGct-3′ (SEQ ID NO: 4838)3′-UUUGUACGUGUAAAACAUGUAACACGA-5′ (SEQ ID NO: 4595) KRAS-5164 Target:5′-AAACATGCACATTTTGTACATTGTGCT-3′ (SEQ ID NO: 5081)  5′-UGCACAUUUUGUACAUUGUGCUUtc-3′ (SEQ ID NO: 4839)3′-GUACGUGUAAAACAUGUAACACGAAAG-5′ (SEQ ID NO: 4596) KRAS-5167 Target:5′-CATGCACATTTTGTACATTGTGCTTTC-3′ (SEQ ID NO: 5082)  5′-GCACAUUUUGUACAUUGUGCUUUct-3′ (SEQ ID NO: 4840)3′-UACGUGUAAAACAUGUAACACGAAAGA-5′ (SEQ ID NO: 4597) KRAS-5168 Target:5′-ATGCACATTTTGTACATTGTGCTTTCT-3′ (SEQ ID NO: 5083)  5′-CACAUUUUGUACAUUGUGCUUUCtt-3′ (SEQ ID NO: 4841)3′-ACGUGUAAAACAUGUAACACGAAAGAA-5′ (SEQ ID NO: 4598) KRAS-5169 Target:5′-TGCACATTTTGTACATTGTGCTTTCTT-3′ (SEQ ID NO: 5084)  5′-ACAUUUUGUACAUUGUGCUUUCUtt-3′ (SEQ ID NO: 4842)3′-CGUGUAAAACAUGUAACACGAAAGAAA-5′ (SEQ ID NO: 4599) KRAS-5170 Target:5′-GCACATTTTGTACATTGTGCTTTCTTT-3′ (SEQ ID NO: 5085)  5′-CAUUUUGUACAUUGUGCUUUCUUtt-3′ (SEQ ID NO: 4843)3′-GUGUAAAACAUGUAACACGAAAGAAAA-5′ (SEQ ID NO: 4600) KRAS-5171 Target:5′-CACATTTTGTACATTGTGCTTTCTTTT-3′ (SEQ ID NO: 5086)  5′-AUUUUGUACAUUGUGCUUUCUUUtg-3′ (SEQ ID NO: 4844)3′-UGUAAAACAUGUAACACGAAAGAAAAC-5′ (SEQ ID NO: 4601) KRAS-5172 Target:5′-ACATTTTGTACATTGTGCTTTCTTTTG-3′ (SEQ ID NO: 5087)  5′-UUUUGUACAUUGUGCUUUCUUUUgt-3′ (SEQ ID NO: 4845)3′-GUAAAACAUGUAACACGAAAGAAAACA-5′ (SEQ ID NO: 4602) KRAS-5173 Target:5′-CATTTTGTACATTGTGCTTTCTTTTGT-3′ (SEQ ID NO: 5088)  5′-UGGGACAUAUGCAGUGUGAUCCAgt-3′ (SEQ ID NO: 4846)3′-ACACCCUGUAUACGUCACACUAGGUCA-5′ (SEQ ID NO: 4603) KRAS-5197 Target:5′-TGTGGGACATATGCAGTGTGATCCAGT-3′ (SEQ ID NO: 5089)  5′-GGGACAUAUGCAGUGUGAUCCAGtt-3′ (SEQ ID NO: 4847)3′-CACCCUGUAUACGUCACACUAGGUCAA-5′ (SEQ ID NO: 4604) KRAS-5198 Target:5′-GTGGGACATATGCAGTGTGATCCAGTT-3′ (SEQ ID NO: 5090)  5′-GGACAUAUGCAGUGUGAUCCAGUtg-3′ (SEQ ID NO: 4848)3′-ACCCUGUAUACGUCACACUAGGUCAAC-5′ (SEQ ID NO: 4605) KRAS-5199 Target:5′-TGGGACATATGCAGTGTGATCCAGTTG-3′ (SEQ ID NO: 5091)  5′-GACAUAUGCAGUGUGAUCCAGUUgt-3′ (SEQ ID NO: 4849)3′-CCCUGUAUACGUCACACUAGGUCAACA-5′ (SEQ ID NO: 4606) KRAS-5200 Target:5′-GGGACATATGCAGTGTGATCCAGTTGT-3′ (SEQ ID NO: 5092)  5′-ACAUAUGCAGUGUGAUCCAGUUGtt-3′ (SEQ ID NO: 4850)3′-CCUGUAUACGUCACACUAGGUCAACAA-5′ (SEQ ID NO: 4607) KRAS-5201 Target:5′-GGACATATGCAGTGTGATCCAGTTGTT-3′ (SEQ ID NO: 5093)  5′-CAUAUGCAGUGUGAUCCAGUUGUtt-3′ (SEQ ID NO: 4851)3′-CUGUAUACGUCACACUAGGUCAACAAA-5′ (SEQ ID NO: 4608) KRAS-5202 Target:5′-GACATATGCAGTGTGATCCAGTTGTTT-3′ (SEQ ID NO: 5094)  5′-AUAUGCAGUGUGAUCCAGUUGUUtt-3′ (SEQ ID NO: 4852)3′-UGUAUACGUCACACUAGGUCAACAAAA-5′ (SEQ ID NO: 4609) KRAS-5203 Target:5′-ACATATGCAGTGTGATCCAGTTGTTTT-3′ (SEQ ID NO: 5095)  5′-UAUGCAGUGUGAUCCAGUUGUUUtc-3′ (SEQ ID NO: 4853)3′-GUAUACGUCACACUAGGUCAACAAAAG-5′ (SEQ ID NO: 4610) KRAS-5204 Target:5′-CATATGCAGTGTGATCCAGTTGTTTTC-3′ (SEQ ID NO: 5096)  5′-AUGCAGUGUGAUCCAGUUGUUUUcc-3′ (SEQ ID NO: 4854)3′-UAUACGUCACACUAGGUCAACAAAAGG-5′ (SEQ ID NO: 4611) KRAS-5205 Target:5′-ATATGCAGTGTGATCCAGTTGTTTTCC-3′ (SEQ ID NO: 5097)  5′-AGUGUGAUCCAGUUGUUUUCCAUca-3′ (SEQ ID NO: 4855)3′-CGUCACACUAGGUCAACAAAAGGUAGU-5′ (SEQ ID NO: 4612) KRAS-5209 Target:5′-GCAGTGTGATCCAGTTGTTTTCCATCA-3′ (SEQ ID NO: 5098)  5′-GUGUGAUCCAGUUGUUUUCCAUCat-3′ (SEQ ID NO: 4856)3′-GUCACACUAGGUCAACAAAAGGUAGUA-5′ (SEQ ID NO: 4613) KRAS-5210 Target:5′-CAGTGTGATCCAGTTGTTTTCCATCAT-3′ (SEQ ID NO: 5099)  5′-UGUGAUCCAGUUGUUUUCCAUCAtt-3′ (SEQ ID NO: 4857)3′-UCACACUAGGUCAACAAAAGGUAGUAA-5′ (SEQ ID NO: 4614) KRAS-5211 Target:5′-AGTGTGATCCAGTTGTTTTCCATCATT-3′ (SEQ ID NO: 5100)  5′-GUGAUCCAGUUGUUUUCCAUCAUtt-3′ (SEQ ID NO: 4858)3′-CACACUAGGUCAACAAAAGGUAGUAAA-5′ (SEQ ID NO: 4615) KRAS-5212 Target:5′-GTGTGATCCAGTTGTTTTCCATCATTT-3′ (SEQ ID NO: 5101)  5′-UGAUCCAGUUGUUUUCCAUCAUUtg-3′ (SEQ ID NO: 4859)3′-ACACUAGGUCAACAAAAGGUAGUAAAC-5′ (SEQ ID NO: 4616) KRAS-5213 Target:5′-TGTGATCCAGTTGTTTTCCATCATTTG-3′ (SEQ ID NO: 5102)  5′-GAUCCAGUUGUUUUCCAUCAUUUgg-3′ (SEQ ID NO: 4860)3′-CACUAGGUCAACAAAAGGUAGUAAACC-5′ (SEQ ID NO: 4617) KRAS-5214 Target:5′-GTGATCCAGTTGTTTTCCATCATTTGG-3′ (SEQ ID NO: 5103)  5′-UUUGGUUGCGCUGACCUAGGAAUgt-3′ (SEQ ID NO: 4861)3′-GUAAACCAACGCGACUGGAUCCUUACA-5′ (SEQ ID NO: 4618) KRAS-5234 Target:5′-CATTTGGTTGCGCTGACCTAGGAATGT-3′ (SEQ ID NO: 5104)  5′-UUGGUUGCGCUGACCUAGGAAUGtt-3′ (SEQ ID NO: 4862)3′-UAAACCAACGCGACUGGAUCCUUACAA-5′ (SEQ ID NO: 4619) KRAS-5235 Target:5′-ATTTGGTTGCGCTGACCTAGGAATGTT-3′ (SEQ ID NO: 5105)  5′-GGAAUGUUGGUCAUAUCAAACAUta-3′ (SEQ ID NO: 4863)3′-AUCCUUACAACCAGUAUAGUUUGUAAU-5′ (SEQ ID NO: 4620) KRAS-5252 Target:5′-TAGGAATGTTGGTCATATCAAACATTA-3′ (SEQ ID NO: 5106)  5′-GAAUGUUGGUCAUAUCAAACAUUaa-3′ (SEQ ID NO: 4864)3′-UCCUUACAACCAGUAUAGUUUGUAAUU-5′ (SEQ ID NO: 4621) KRAS-5253 Target:5′-AGGAATGTTGGTCATATCAAACATTAA-3′ (SEQ ID NO: 5107)  5′-AAUGUUGGUCAUAUCAAACAUUAaa-3′ (SEQ ID NO: 4865)3′-CCUUACAACCAGUAUAGUUUGUAAUUU-5′ (SEQ ID NO: 4622) KRAS-5254 Target:5′-GGAATGTTGGTCATATCAAACATTAAA-3′ (SEQ ID NO: 5108)  5′-AUGUUGGUCAUAUCAAACAUUAAaa-3′ (SEQ ID NO: 4866)3′-CUUACAACCAGUAUAGUUUGUAAUUUU-5′ (SEQ ID NO: 4623) KRAS-5255 Target:5′-GAATGTTGGTCATATCAAACATTAAAA-3′ (SEQ ID NO: 5109)  5′-UGUUGGUCAUAUCAAACAUUAAAaa-3′ (SEQ ID NO: 4867)3′-UUACAACCAGUAUAGUUUGUAAUUUUU-5′ (SEQ ID NO: 4624) KRAS-5256 Target:5′-AATGTTGGTCATATCAAACATTAAAAA-3′ (SEQ ID NO: 5110)  5′-GUUGGUCAUAUCAAACAUUAAAAat-3′ (SEQ ID NO: 4868)3′-UACAACCAGUAUAGUUUGUAAUUUUUA-5′ (SEQ ID NO: 4625) KRAS-5257 Target:5′-ATGTTGGTCATATCAAACATTAAAAAT-3′ (SEQ ID NO: 5111)  5′-UUGGUCAUAUCAAACAUUAAAAAtg-3′ (SEQ ID NO: 4869)3′-ACAACCAGUAUAGUUUGUAAUUUUUAC-5′ (SEQ ID NO: 4626) KRAS-5258 Target:5′-TGTTGGTCATATCAAACATTAAAAATG-3′ (SEQ ID NO: 5112)  5′-UGGUCAUAUCAAACAUUAAAAAUga-3′ (SEQ ID NO: 4870)3′-CAACCAGUAUAGUUUGUAAUUUUUACU-5′ (SEQ ID NO: 4627) KRAS-5259 Target:5′-GTTGGTCATATCAAACATTAAAAATGA-3′ (SEQ ID NO: 5113)  5′GGUCAUAUCAAACAUUAAAAAUGac.73′ (SEQ ID NO: 4871)3′-AACCAGUAUAGUUUGUAAUUUUUACUG-5′ (SEQ ID NO: 4628) KRAS-5260 Target:5′-TTGGTCATATCAAACATTAAAAATGAC-3′ (SEQ ID NO: 5114)  5′-AAAUUAACUUUUAAAUGUUUAUAgg-3′ (SEQ ID NO: 4872)3′-ACUUUAAUUGAAAAUUUACAAAUAUCC-5′ (SEQ ID NO: 4629) KRAS-5299 Target:5′-TGAAATTAACTTTTAAATGTTTATAGG-3′ (SEQ ID NO: 5115)  5′-AAUUAACUUUUAAAUGUUUAUAGga-3′ (SEQ ID NO: 4873)3′-CUUUAAUUGAAAAUUUACAAAUAUCCU-5′ (SEQ ID NO: 4630) KRAS-5300 Target:5′-GAAATTAACTTTTAAATGTTTATAGGA-3′ (SEQ ID NO: 5116)  5′-AACUUUUAAAUGUUUAUAGGAGUat-3′ (SEQ ID NO: 4874)3′-AAUUGAAAAUUUACAAAUAUCCUCAUA-5′ (SEQ ID NO: 4631) KRAS-5304 Target:5′-TTAACTTTTAAATGTTTATAGGAGTAT-3′ (SEQ ID NO: 5117)  5′-ACUUUUAAAUGUUUAUAGGAGUAtg-3′ (SEQ ID NO: 4875)3′-AUUGAAAAUUUACAAAUAUCCUCAUAC-5′ (SEQ ID NO: 4632) KRAS-5305 Target:5′-TAACTTTTAAATGTTTATAGGAGTATG-3′ (SEQ ID NO: 5118)  5′-CUUUUAAAUGUUUAUAGGAGUAUgt-3′ (SEQ ID NO: 4876)3′-UUGAAAAUUUACAAAUAUCCUCAUACA-5′ (SEQ ID NO: 4633) KRAS-5306 Target:5′-AACTTTTAAATGTTTATAGGAGTATGT-3′ (SEQ ID NO: 5119)  5′-UUUUAAAUGUUUAUAGGAGUAUGtg-3′ (SEQ ID NO: 4877)3′-UGAAAAUUUACAAAUAUCCUCAUACAC-5′ (SEQ ID NO: 4634) KRAS-5307 Target:5′-ACTTTTAAATGTTTATAGGAGTATGTG-3′ (SEQ ID NO: 5120)  5′-UUUAAAUGUUUAUAGGAGUAUGUgc-3′ (SEQ ID NO: 4878)3′-GAAAAUUUACAAAUAUCCUCAUACACG-5′ (SEQ ID NO: 4635) KRAS-5308 Target:5′-CTTTTAAATGTTTATAGGAGTATGTGC-3′ (SEQ ID NO: 5121)  5′-UUAAAUGUUUAUAGGAGUAUGUGct-3′ (SEQ ID NO: 4879)3′-AAAAUUUACAAAUAUCCUCAUACACGA-5′ (SEQ ID NO: 4636) KRAS-5309 Target:5′-TTTTAAATGTTTATAGGAGTATGTGCT-3′ (SEQ ID NO: 5122)  5′-AAAUUUGUAAUAUUUUUGUCAUGaa-3′ (SEQ ID NO: 4880)3′-AUUUUAAACAUUAUAAAAACAGUACUU-5′ (SEQ ID NO: 4637) KRAS-5347 Target:5′-TAAAATTTGTAATATTTTTGTCATGAA-3′ (SEQ ID NO: 5123)  5′-AAUUUGUAAUAUUUUUGUCAUGAac-3′ (SEQ ID NO: 4881)3′-UUUUAAACAUUAUAAAAACAGUACUUG-5′ (SEQ ID NO: 4638) KRAS-5348 Target:5′-AAAATTTGTAATATTTTTGTCATGAAC-3′ (SEQ ID NO: 5124)  5′-AUUUGUAAUAUUUUUGUCAUGAAct-3′ (SEQ ID NO: 4882)3′-UUUAAACAUUAUAAAAACAGUACUUGA-5′ (SEQ ID NO: 4639) KRAS-5349 Target:5′-AAATTTGTAATATTTTTGTCATGAACT-3′ (SEQ ID NO: 5125)  5′-UUUGUAAUAU6UUUGUCAUGAACtg-3′ (SEQ ID NO: 4883)3′-UUAAACAUUAUAAAAACAGUACUUGAC-5′ (SEQ ID NO: 4640) KRAS-5350 Target:5′-AATTTGTAATATTTTTGTCATGAACTG-3′ (SEQ ID NO: 5126)  5′-UUGUAAUAUUUUUGUCAUGAACUgt-3′ (SEQ ID NO: 4884)3′-UAAACAUUAUAAAAACAGUACUUGACA-5′ (SEQ ID NO: 4641) KRAS-5351 Target:5′-ATTTGTAATATTTTTGTCATGAACTGT-3′ (SEQ ID NO: 5127)  5′-UGUAAUAUUUUUGUCAUGAACUGta-3′ (SEQ ID NO: 4885)3′-AAACAUUAUAAAAACAGUACUUGACAU-5′ (SEQ ID NO: 4642) KRAS-5352 Target:5′-TTTGTAATATTTTTGTCATGAACTGTA-3′ (SEQ ID NO: 5128)  5′-GUAAUAUUUUUGUCAUGAACUGUac-3′ (SEQ ID NO: 4886)3′-AACAUUAUAAAAACAGUACUUGACAUG-5′ (SEQ ID NO: 4643) KRAS-5353 Target:5′-TTGTAATATTTTTGTCATGAACTGTAC-3′ (SEQ ID NO: 5129)  5′-UAAUAUUUUUGUCAUGAACUGUAct-3′ (SEQ ID NO: 4887)3′-ACAUUAUAAAAACAGUACUUGACAUGA-5′ (SEQ ID NO: 4644) KRAS-5354 Target:5′-TGTAATATTTTTGTCATGAACTGTACT-3′ (SEQ ID NO: 5130)  5′-AUUGUAAUGUAAUAAAAAUAGUUac-3′ (SEQ ID NO: 4888)3′-AAUAACAUUACAUUAUUUUUAUCAAUG-5′ (SEQ ID NO: 4645) KRAS-5389 Target:5′-TTATTGTAATGTAATAAAAATAGTTAC-3′ (SEQ ID NO: 5131)  5′-UUGUAAUGUAAUAAAAAUAGUUAca-3′ (SEQ ID NO: 4889)3′-AUAACAUUACAUUAUUUUUAUCAAUGU-5′ (SEQ ID NO: 4646) KRAS-5390 Target:5′-TATTGTAATGTAATAAAAATAGTTACA-3′ (SEQ ID NO: 5132)  5′-UGUAAUGUAAUAAAAAUAGUUACag-3′ (SEQ ID NO: 4890)3′-UAACAUUACAUUAUUUUUAUCAAUGUC-5′ (SEQ ID NO: 4647) KRAS-5391 Target:5′-ATTGTAATGTAATAAAAATAGTTACAG-3′ (SEQ ID NO: 5133)  5′-GUAAUGUAAUAAAAAUAGUUACAgt-3′ (SEQ ID NO: 4891)3′-AACAUUACAUUAUUUUUAUCAAUGUCA-5′ (SEQ ID NO: 4648) KRAS-5392 Target:5′-TTGTAATGTAATAAAAATAGTTACAGT-3′ (SEQ ID NO: 5134)  5′-UAAUGUAAUAAAAAUAGUUACAGtg-3′ (SEQ ID NO: 4892)3′-ACAUUACAUUAUUUUUAUCAAUGUCAC-5′ (SEQ ID NO: 4649) KRAS-5393 Target:5′-TGTAATGTAATAAAAATAGTTACAGTG-3′ (SEQ ID NO: 5135)

As in Tables 2-5 above, underlined nucleotide residues of above Table 8indicate 2′-O-methyl modified residues; ribonucleotide residues areshown as UPPER CASE, while deoxyribonucleotide residues are shown aslower case.

Assay of the above 243 KRAS targeting DsiRNAs in human HeLa and mouseHepa 1-6 cells at 1 nM revealed the following KRAS inhibitoryefficacies, presented in Table 9. KRAS levels were determined usingpaired qPCR assays positioned at distinct locations within the KRAStranscript (for HeLa cell experiments, qPCR assays are indicated as “HsKRAS 268-385 (MAX)” and “Hs KRAS 3393-3516 (FAM)”; for Hepa 1-6 cellexperiments, such assays are indicated as “Mm KRAS 279-390 (MAX)” and“Mm KRAS1064-1161 (FAM)”).

TABLE 9 KRAS Inhibitory Efficacy of Table 8 DsiRNAs Assayed at 1 nM inHuman HeLa and Mouse Hepa 1-6 Cells Human - HeLa Mouse - Hepa 1-6Normalized HPRT/SFRS9; vs NC1, NC5, NC7 Normalized HPRT/Rpl23; vs NC1,NC5, NC7 Assay: Hs KRAS Assay: Hs KRAS Assay: Mm KRAS Assay: Mm KRAS268-385 (MAX) 3393-3516 (FAM) 279-390 (MAX) 1064-1161 (FAM) Hs Mm % % %% % % % % Name Loc Loc nM Left Ave Error Left Ave Error Left Ave ErrorLeft Ave Error Hs|NM_033360.2|166 166 179 1 16.9 5.8 18.8 8.6 43.4 1.047.5 5.1 Hs|NM_033360.2|167 167 180 1 22.4 6.8 28.5 5.4 54.1 5.0 53.02.7 Hs|NM_033360.2|168 168 181 1 12.0 3.0 20.1 4.2 47.9 3.9 43.7 2.6Hs|NM_033360.2|169 169 182 1 12.4 1.6 21.7 2.6 38.7 3.0 31.5 1.5Hs|NM_033360.2|204 204 217 1 92.7 11.5 107.2 11.4 109.3 5.4 96.7 13.5Hs|NM_033360.2|205 205 218 1 105.3 7.7 127.7 5.6 108.2 3.3 94.9 4.5Hs|NM_033360.2|206 206 219 1 67.1 4.7 80.9 10.9 71.9 1.6 59.3 2.6Hs|NM_033360.2|207 207 220 1 46.0 N/A 62.7 N/A 63.1 5.9 55.8 5.8Hs|NM_033360.2|208 208 221 1 54.6 10.4 47.3 14.6 53.4 1.9 59.0 1.9Hs|NM_033360.2|209 209 222 1 83.2 3.4 90.4 4.2 89.5 6.1 90.8 4.1Hs|NM_033360.2|210 210 223 1 78.1 5.2 89.9 5.6 96.4 1.3 88.2 2.9Hs|NM_033360.2|241 241 254 1 14.5 10.8 22.4 4.9 42.6 5.9 36.4 3.8Hs|NM_033360.2|313 313 326 1 32.2 6.7 51.5 3.7 63.1 5.6 53.8 4.4Hs|NM_033360.2|314 314 327 1 34.2 6.8 46.6 10.0 66.6 3.3 56.2 3.6Hs|NM_033360.2|318 318 331 1 10.9 9.2 27.6 6.8 43.0 10.4 30.3 9.0Hs|NM_033360.2|328 328 341 1 25.1 5.8 28.1 6.8 47.6 6.4 36.2 3.1Hs|NM_033360.2|330 330 343 1 10.3 5.5 17.2 8.0 26.2 3.5 25.0 6.1Hs|NM_033360.2|331 331 344 1 15.1 6.2 27.0 7.4 44.0 6.0 37.2 6.0Hs|NM_033360.2|332 332 345 1 20.8 9.6 40.2 5.8 45.7 11.0 38.0 5.4Hs|NM_033360.2|333 333 346 1 19.4 3.8 38.6 3.8 47.2 5.9 36.3 3.4Hs|NM_033360.2|334 334 347 1 7.6 5.2 29.6 3.2 35.6 3.3 28.8 5.8Hs|NM_033360.2|335 335 348 1 28.0 10.3 41.2 16.6 72.8 6.5 55.2 10.7Hs|NM_033360.2|336 336 349 1 16.5 9.3 31.9 1.1 57.0 4.0 41.7 2.6Hs|NM_033360.2|352 352 365 1 15.7 7.0 29.5 2.2 64.9 6.1 43.3 4.6Hs|NM_033360.2|353 353 366 1 7.2 5.6 26.1 7.0 35.7 10.8 26.3 5.7Hs|NM_033360.2|354 354 367 1 15.3 3.8 42.4 3.9 57.1 1.6 41.8 2.6Hs|NM_033360.2|364 364 377 1 31.3 1.8 44.3 6.2 57.1 2.6 47.8 3.9Hs|NM_033360.2|365 365 378 1 10.0 13.3 23.1 5.8 38.8 2.1 28.3 6.4Hs|NM_033360.2|366 366 379 1 11.1 8.0 27.1 8.3 44.3 5.4 28.7 2.5Hs|NM_033360.2|367 367 380 1 14.6 11.3 37.3 8.7 58.4 3.2 34.5 5.2Hs|NM_033360.2|368 368 381 1 54.4 2.6 81.7 11.4 113.2 9.3 81.6 8.6Hs|NM_033360.2|369 369 382 1 73.5 N/A 83.8 N/A 104.8 27.8 90.3 9.3Hs|NM_033360.2|370 370 383 1 56.7 3.8 61.9 5.0 69.0 3.2 66.4 1.8Hs|NM_033360.2|371 371 384 1 12.7 9.4 22.7 8.8 47.4 2.8 42.2 3.8Hs|NM_033360.2|372 372 385 1 26.6 7.9 63.7 12.0 75.0 3.0 49.2 4.3Hs|NM_033360.2|420 420 433 1 11.8 11.9 21.5 10.5 39.9 5.0 31.5 4.1Hs|NM_033360.2|421 421 434 1 40.7 10.6 55.4 13.0 72.0 8.5 59.6 7.7Hs|NM_033360.2|422 422 435 1 18.1 13.3 30.2 18.8 41.8 5.8 32.1 11.0Hs|NM_033360.2|423 423 436 1 18.6 14.2 27.2 13.4 52.3 7.6 40.5 5.0Hs|NM_033360.2|424 424 437 1 10.8 14.3 24.3 7.7 31.6 12.7 24.4 3.0Hs|NM_033360.2|425 425 438 1 10.2 6.6 16.7 10.6 26.1 4.5 27.0 4.9Hs|NM_033360.2|426 426 439 1 10.4 13.1 22.7 10.6 32.0 3.9 28.9 4.5Hs|NM_033360.2|436 436 449 1 9.6 11.4 19.2 6.5 37.6 5.9 33.4 4.7Hs|NM_033360.2|437 437 450 1 22.8 5.2 32.9 5.2 53.6 5.4 46.2 5.2Hs|NM_033360.2|438 438 451 1 11.0 9.4 18.6 3.9 34.5 1.7 26.7 3.8Hs|NM_033360.2|439 439 452 1 14.0 15.4 18.6 11.3 40.5 4.6 28.6 4.5Hs|NM_033360.2|440 440 453 1 12.6 2.2 22.8 4.4 43.1 4.5 33.9 5.3Hs|NM_033360.2|441 441 454 1 10.9 11.6 20.7 8.9 40.1 8.9 28.7 2.7Hs|NM_033360.2|442 442 455 1 16.8 2.1 23.4 4.0 37.1 5.9 37.3 9.2Hs|NM_033360.2|443 443 456 1 17.2 8.9 26.9 6.6 47.5 2.4 45.8 3.7Hs|NM_033360.2|444 444 457 1 13.3 6.7 20.0 1.9 35.1 3.2 32.3 2.7Hs|NM_033360.2|454 454 467 1 8.4 6.5 18.2 8.4 42.5 4.3 33.7 4.4Hs|NM_033360.2|455 455 468 1 11.7 7.0 19.9 5.0 44.2 10.4 41.5 3.1Hs|NM_033360.2|456 456 469 1 13.0 6.4 24.6 1.6 42.8 3.4 30.9 6.4Hs|NM_033360.2|457 457 470 1 10.8 7.7 21.4 1.3 43.4 8.2 32.7 3.4Hs|NM_033360.2|458 458 471 1 13.7 3.5 15.2 19.2 37.0 2.9 28.6 8.7Hs|NM_033360.2|459 459 472 1 11.5 9.3 14.8 2.4 31.8 1.5 32.1 3.0Hs|NM_033360.2|460 460 473 1 5.0 12.7 11.3 9.3 37.2 0.2 32.7 2.1Hs|NM_033360.2|461 461 474 1 7.6 9.6 12.7 8.5 31.6 1.5 28.1 2.9Hs|NM_033360.2|462 462 475 1 6.8 14.3 14.1 7.6 33.3 3.2 27.8 3.2Hs|NM_033360.2|508 508 h534 1 16.8 6.6 22.8 14.8 102.3 9.0 79.5 8.1Hs|NM_033360.2|531 531 544 1 31.4 2.1 36.0 5.0 63.9 3.4 62.9 20.1Hs|NM_033360.2|532 532 545 1 16.2 7.1 22.6 7.5 43.0 4.9 33.2 4.2Hs|NM_033360.2|534 534 547 1 15.5 3.7 20.7 3.8 44.8 4.4 35.1 8.6Hs|NM_033360.2|586 586 h612 1 9.4 4.3 11.8 4.9 86.3 7.2 91.3 7.9Hs|NM_033360.2|587 587 h613 1 20.1 4.6 30.6 4.5 102.6 1.7 103.2 2.4Hs|NM_033360.2|588 588 h614 1 22.0 9.3 23.9 13.9 104.1 14.0 104.4 9.8Hs|NM_033360.2|763 763 652 1 16.4 6.0 20.3 4.8 42.6 1.6 37.9 2.1Hs|NM_033360.2|764 764 653 1 15.9 4.2 21.3 3.4 36.8 6.2 32.9 5.4Hs|NM_033360.2|784 784 673 1 8.5 2.8 13.3 5.9 31.7 15.4 25.2 17.6Hs|NM_033360.2|794 794 683 1 11.4 8.4 18.1 7.7 31.9 9.8 24.8 7.9Hs|NM_033360.2|795 795 684 1 10.4 6.7 13.9 2.5 27.7 7.0 23.2 7.2Hs|NM_033360.2|796 796 685 1 30.5 2.0 33.2 2.0 25.3 N/A 37.8 N/AHs|NM_033360.2|797 797 686 1 12.7 2.3 20.9 3.5 23.2 1.8 29.6 2.4Hs|NM_033360.2|798 798 687 1 11.0 11.3 28.2 9.3 27.5 4.1 35.4 2.2Hs|NM_033360.2|799 799 688 1 8.7 8.4 16.7 4.7 119.5 1.7 100.7 4.1Hs|NM_033360.2|800 800 689 1 13.6 7.4 19.3 6.7 144.8 21.8 105.1 7.8Hs|NM_033360.2|801 801 690 1 28.5 5.8 40.8 5.8 37.0 5.3 40.8 4.9Hs|NM_033360.2|802 802 691 1 13.9 4.6 37.2 2.0 152.1 2.9 124.7 6.4Hs|NM_033360.2|908 908 h810 1 21.4 12.6 17.4 13.1 130.7 7.5 117.6 5.3Hs|NM_033360.2|909 909 h811 1 11.6 2.0 9.0 7.7 90.4 2.6 100.0 2.6Hs|NM_033360.2|920 920 807 1 15.9 7.1 14.1 11.6 110.8 4.6 107.1 4.4Hs|NM_033360.2|921 921 808 1 15.5 9.3 18.4 11.9 118.2 11.7 108.9 9.8Hs|NM_033360.2|922 922 809 1 14.3 8.5 13.2 6.5 135.9 4.6 110.9 6.5Hs|NM_033360.2|923 923 810 1 17.3 8.7 12.6 12.0 129.0 2.2 106.2 5.2Hs|NM_033360.2|924 924 811 1 19.6 3.1 20.7 8.8 111.5 8.5 100.9 7.4Hs|NM_033360.2|925 925 812 1 22.2 6.0 28.8 8.6 115.3 3.2 99.8 1.7Hs|NM_033360.2|926 926 813 1 21.2 23.3 27.7 8.6 114.7 18.4 124.1 5.8Hs|NM_033360.2|927 927 814 1 15.6 10.3 10.7 1.7 99.2 7.3 103.9 6.7Hs|NM_033360.2|928 928 815 1 26.1 4.7 21.6 3.0 118.0 4.4 111.7 5.3Hs|NM_033360.2|938 938 825 1 26.0 7.2 22.1 17.7 106.2 9.8 107.7 6.9Hs|NM_033360.2|939 939 826 1 13.9 3.9 13.9 11.1 30.9 8.9 32.2 10.9Hs|NM_033360.2|940 940 827 1 19.7 6.4 22.2 8.2 28.0 7.0 34.2 4.8Hs|NM_033360.2|941 941 828 1 13.8 9.7 15.0 12.2 26.0 17.1 29.5 15.2Hs|NM_033360.2|942 942 829 1 15.9 2.8 18.3 7.0 50.1 5.8 48.2 2.3Hs|NM_033360.2|943 943 830 1 22.8 N/A 25.1 N/A 128.1 3.3 113.3 7.6Hs|NM_033360.2|944 944 831 1 22.1 11.4 15.7 7.2 45.5 3.8 44.1 2.3Hs|NM_033360.2|945 945 832 1 15.6 8.3 12.7 8.1 32.7 2.2 29.4 2.8Hs|NM_033360.2|946 946 833 1 13.7 7.4 12.9 2.5 29.7 2.3 25.8 4.4Hs|NM_033360.2|1010 1010 h912 1 20.6 3.6 22.6 1.6 22.8 4.5 17.2 3.7Hs|NM_033360.2|1012 1012 h914 1 24.4 N/A 28.3 N/A 29.9 8.6 23.3 9.1Hs|NM_033360.2|1045 1045 936 1 18.3 10.7 19.3 6.7 52.5 4.3 42.0 3.1Hs|NM_033360.2|1197 1197 h1099 1 12.2 8.9 11.6 8.4 30.4 6.3 24.5 3.1Hs|NM_033360.2|1198 1198 h1100 1 17.3 12.1 12.8 5.8 138.8 11.4 114.918.5 Hs|NM_033360.2|1230 1230 h1132 1 18.4 12.0 11.6 12.5 75.1 1.5 83.51.2 Hs|NM_033360.2|1231 1231 h1133 1 16.7 7.1 12.7 5.0 20.5 8.2 26.0 6.3Hs|NM_033360.2|1234 1234 h1136 1 21.9 4.0 16.4 3.0 24.3 8.6 26.6 10.3Hs|NM_033360.2|1249 1249 h1151 1 20.1 1.5 12.0 1.8 20.9 3.9 22.9 6.1Hs|NM_033360.2|1250 1250 h1152 1 26.2 6.7 21.0 7.3 27.4 5.8 30.8 7.0Hs|NM_033360.2|1287 1287 h1189 1 44.3 6.8 40.1 4.3 30.1 6.5 31.2 6.6Hs|NM_033360.2|1527 1527 h1429 1 34.0 2.7 17.3 5.7 40.8 4.1 41.0 3.5Hs|NM_033360.2|1533 1533 h1435 1 25.6 17.6 15.4 4.8 33.7 8.8 49.1 21.9Hs|NM_033360.2|1540 1540 h1442 1 25.6 6.2 11.4 5.6 18.3 15.2 26.9 11.0Hs|NM_033360.2|1541 1541 h1443 1 25.2 4.7 9.8 6.2 40.3 9.4 48.6 6.5Hs|NM_033360.2|1542 1542 h1444 1 41.4 5.6 32.1 7.2 32.0 7.8 34.4 15.7Hs|NM_033360.2|1583 1583 1435 1 22.5 4.3 13.3 2.1 23.3 9.6 25.1 9.6Hs|NM_033360.2|1584 1584 1436 1 32.7 2.0 24.5 3.1 28.1 4.1 29.0 4.1Hs|NM_033360.2|1585 1585 1437 1 32.9 7.8 22.2 9.7 16.6 10.1 18.5 11.7Hs|NM_033360.2|1586 1586 1438 1 52.5 2.5 36.2 6.1 23.8 7.2 26.0 3.6Hs|NM_033360.2|1597 1597 h1499 1 38.2 N/A 36.1 N/A 29.3 7.6 31.1 3.9Hs|NM_033360.2|1606 1606 h1508 1 45.4 5.0 28.5 4.2 110.9 2.9 119.3 2.0Hs|NM_033360.2|1630 1630 1471 1 23.2 8.0 16.5 8.1 26.8 7.3 34.7 4.8Hs|NM_033360.2|1631 1631 1472 1 35.4 5.2 23.6 5.7 37.1 4.6 44.0 4.2Hs|NM_033360.2|1632 1632 1473 1 26.9 2.3 20.0 1.6 29.8 4.3 32.8 1.8Hs|NM_033360.2|1633 1633 1474 1 25.4 5.8 24.4 13.4 31.6 3.4 38.9 7.6Hs|NM_033360.2|1634 1634 1475 1 27.8 3.5 20.3 3.6 28.5 5.9 31.6 5.3Hs|NM_033360.2|1635 1635 1476 1 29.8 0.4 23.2 3.4 37.4 4.9 38.7 4.7Hs|NM_033360.2|1636 1636 1477 1 51.0 22.3 38.9 11.1 63.5 11.6 65.0 6.7Hs|NM_033360.2|1637 1637 1478 1 31.5 7.4 14.0 11.9 28.2 6.0 37.3 6.0Hs|NM_033360.2|1638 1638 1479 1 31.1 1.4 27.4 2.5 44.1 8.9 51.8 3.0Hs|NM_033360.2|1639 1639 1480 1 47.7 4.9 26.0 7.7 57.0 3.2 60.9 2.6Hs|NM_033360.2|1640 1640 1481 1 35.1 3.7 17.7 4.2 62.1 2.6 59.9 3.7Hs|NM_033360.2|1736 1736 h1638 1 30.9 3.9 20.2 7.0 133.0 2.5 110.1 1.7Hs|NM_033360.2|1741 1741 h1643 1 25.2 9.1 12.4 7.0 126.6 0.8 111.7 4.0Hs|NM_033360.2|1742 1742 h1644 1 18.5 4.4 11.5 6.7 130.1 4.7 106.9 4.9Hs|NM_033360.2|1753 1753 h1655 1 33.4 15.2 29.9 5.2 130.3 3.4 105.4 2.0Hs|NM_033360.2|1754 1754 h1656 1 31.3 5.8 14.6 16.3 93.9 4.7 100.7 4.6Hs|NM_033360.2|1769 1769 h1671 1 25.7 4.9 18.4 11.5 107.6 4.2 129.9 14.3Hs|NM_033360.2|1771 1771 h1673 1 31.4 3.1 13.9 9.0 106.8 12.2 100.8 8.5Hs|NM_033360.2|1772 1772 h1674 1 24.5 3.5 13.2 5.0 111.3 1.0 95.3 2.4Hs|NM_033360.2|1783 1783 h1685 1 31.3 2.2 27.9 11.1 126.6 4.0 109.3 5.4Hs|NM_033360.2|1784 1784 h1686 1 33.6 16.9 22.0 20.9 121.4 15.0 98.015.2 Hs|NM_033360.2|1785 1785 h1687 1 28.2 4.9 19.9 5.0 124.1 2.2 105.55.3 Hs|NM_033360.2|1799 1799 h1701 1 23.0 6.1 15.1 8.4 128.6 1.8 106.82.8 Hs|NM_033360.2|2100 2100 h2002 1 49.7 19.4 34.1 12.1 106.3 5.6 111.42.7 Hs|NM_033360.2|2134 2134 h2036 1 51.2 N/A 35.0 N/A 130.2 3.6 119.64.9 Hs|NM_033360.2|2216 2216 h2118 1 28.4 4.6 13.2 6.6 113.2 2.8 114.02.6 Hs|NM_033360.2|2217 2217 h2119 1 23.6 6.9 11.2 12.2 127.0 1.7 111.91.8 Hs|NM_033360.2|2218 2218 h2120 1 30.7 13.0 16.3 11.9 119.3 2.4 106.37.2 Hs|NM_033360.2|2229 2229 h2131 1 34.4 6.7 16.0 6.5 118.3 3.9 110.18.9 Hs|NM_033360.2|2247 2247 h2149 1 31.2 6.8 18.1 10.6 129.1 6.1 117.76.2 Hs|NM_033360.2|2326 2326 h2228 1 85.1 24.0 118.0 25.0 170.9 9.9151.3 16.5 Hs|NM_033360.2|2327 2327 h2229 1 105.3 4.8 76.9 2.9 106.7 1.1108.6 6.0 Hs|NM_033360.2|2547 2547 2285 1 68.5 3.8 41.4 1.8 98.3 6.3102.5 2.4 Hs|NM_033360.2|2548 2548 2286 1 50.0 6.7 30.2 12.6 85.8 3.280.3 3.1 Hs|NM_033360.2|3741 3741 h3643 1 51.3 21.2 51.4 10.6 110.0 2.597.0 2.8 Hs|NM_033360.2|3746 3746 h3648 1 49.6 10.5 41.6 11.3 129.2 8.7116.8 9.0 Hs|NM_033360.2|3747 3747 h3649 1 42.3 3.9 39.2 3.2 115.5 1.5105.8 1.7 Hs|NM_033360.2|3783 3783 h3685 1 36.2 4.3 31.1 4.7 119.2 2.3108.7 1.3 Hs|NM_033360.2|3784 3784 h3686 1 78.0 20.4 42.2 11.3 141.2 6.9125.0 1.4 Hs|NM_033360.2|3810 3810 h3712 1 56.0 18.6 39.7 19.3 91.2 1.6102.4 2.5 Hs|NM_033360.2|4396 4396 3584 1 58.6 2.6 48.2 2.0 88.8 3.194.0 2.2 Hs|NM_033360.2|4447 4447 3633 1 65.1 3.6 49.0 1.9 78.4 9.3 76.49.6 Hs|NM_033360.2|4448 4448 3634 1 41.7 3.1 31.5 8.6 98.3 4.5 86.0 4.2Hs|NM_033360.2|4449 4449 3635 1 57.3 11.0 54.8 10.9 108.2 7.1 92.5 5.0Hs|NM_033360.2|4450 4450 3636 1 55.8 7.5 54.7 6.3 87.6 7.5 75.0 9.4Hs|NM_033360.2|4451 4451 3637 1 76.7 3.5 74.5 3.8 106.1 1.1 88.3 2.3Hs|NM_033360.2|4452 4452 3638 1 56.1 2.1 50.0 4.1 109.4 4.5 94.8 5.9Hs|NM_033360.2|4748 4748 3940 1 54.7 13.7 41.7 10.8 89.1 0.9 97.6 1.0Hs|NM_033360.2|4749 4749 3941 1 52.3 13.5 41.0 9.5 109.1 5.4 109.6 4.0Hs|NM_033360.2|4878 4878 4082 1 63.9 4.5 63.1 5.1 116.7 7.2 104.9 7.1Hs|NM_033360.2|4879 4879 4083 1 43.5 4.1 41.3 2.3 117.8 4.3 102.2 4.2Hs|NM_033360.2|4880 4880 4084 1 64.1 10.5 75.7 12.4 119.1 3.4 100.4 5.1Hs|NM_033360.2|5073 5073 4259 1 36.8 4.0 36.1 7.2 116.2 2.9 106.8 5.9Hs|NM_033360.2|5074 5074 4260 1 45.0 3.6 58.5 3.0 148.1 7.0 129.1 6.9Hs|NM_033360.2|5075 5075 4261 1 48.5 2.6 53.1 6.6 Hs|NM_033360.2|50765076 4262 1 38.4 9.2 29.4 10.6 83.3 N/A 90.7 N/A Hs|NM_033360.2|50775077 4263 1 32.9 6.0 29.1 4.0 91.5 4.8 91.2 3.5 Hs|NM_033360.2|5078 50784264 1 29.7 7.8 28.6 6.7 99.1 2.6 95.2 1.1 Hs|NM_033360.2|5128 5128 43141 56.7 4.6 62.4 5.3 108.3 3.9 91.6 4.6 Hs|NM_033360.2|5129 5129 4315 154.7 5.3 61.6 6.4 110.3 9.6 91.4 9.8 Hs|NM_033360.2|5138 5138 4324 138.2 6.5 42.1 5.6 102.6 6.7 96.3 7.8 Hs|NM_033360.2|5139 5139 4325 131.5 7.1 35.6 9.6 101.4 0.9 89.0 2.7 Hs|NM_033360.2|5140 5140 4326 147.1 21.2 40.8 15.3 114.9 2.9 96.7 5.7 Hs|NM_033360.2|5141 5141 4327 148.6 4.8 41.9 5.7 76.3 2.9 82.6 3.1 Hs|NM_033360.2|5142 5142 4328 1 60.316.7 54.1 17.5 95.4 1.1 90.9 1.4 Hs|NM_033360.2|5143 5143 4329 1 42.27.7 42.5 10.0 89.2 7.9 84.5 4.0 Hs|NM_033360.2|5163 5163 4349 1 109.72.4 94.3 2.1 Hs|NM_033360.2|5164 5164 4350 1 34.5 6.4 37.5 6.1 94.7 3.679.5 5.3 Hs|NM_033360.2|5167 5167 4353 1 49.4 9.8 54.7 11.0 89.9 12.276.5 14.1 Hs|NM_033360.2|5168 5168 4354 1 46.9 2.4 54.3 5.4 111.9 3.895.9 1.5 Hs|NM_033360.2|5169 5169 4355 1 52.8 3.3 52.1 4.9 113.7 3.199.7 2.8 Hs|NM_033360.2|5170 5170 4356 1 42.2 3.2 38.2 1.6 82.6 5.5 84.13.1 Hs|NM_033360.2|5171 5171 4357 1 51.6 5.7 49.4 4.5 86.4 0.6 92.6 1.4Hs|NM_033360.2|5172 5172 4358 1 56.4 6.2 58.8 4.5 81.0 4.0 79.6 3.7Hs|NM_033360.2|5173 5173 4359 1 44.8 8.5 51.5 8.1 89.8 4.2 87.9 5.7Hs|NM_033360.2|5197 5197 4435 1 63.9 19.5 64.1 10.2 90.5 3.8 94.4 5.1Hs|NM_033360.2|5198 5198 4436 1 47.3 5.7 58.2 6.4 91.9 3.3 92.9 6.0Hs|NM_033360.2|5199 5199 4437 1 50.0 4.8 52.7 4.8 114.0 4.9 95.5 3.9Hs|NM_033360.2|5200 5200 4438 1 65.4 N/A 69.3 N/A 98.1 4.0 88.1 1.8Hs|NM_033360.2|5201 5201 4439 1 54.0 4.4 42.2 5.7 89.1 10.4 79.4 6.4Hs|NM_033360.2|5202 5202 4440 1 45.3 4.6 41.8 5.9 109.5 1.8 92.8 3.7Hs|NM_033360.2|5203 5203 4441 1 35.7 5.7 34.5 3.1 102.3 2.7 87.3 2.3Hs|NM_033360.2|5204 5204 4442 1 51.9 5.3 55.8 3.3 107.5 8.5 90.1 8.9Hs|NM_033360.2|5205 5205 4443 1 57.7 24.4 67.9 34.1 105.0 6.0 90.4 4.0Hs|NM_033360.2|5209 5209 4447 1 55.1 7.8 58.9 7.2 121.2 7.6 102.1 6.1Hs|NM_033360.2|5210 5210 4448 1 46.9 8.9 45.0 9.1 106.8 5.0 94.1 3.1Hs|NM_033360.2|5211 5211 4449 1 54.4 11.7 54.8 11.5 107.4 4.8 94.8 6.4Hs|NM_033360.2|5212 5212 4450 1 41.3 7.5 33.3 6.6 101.2 6.8 83.0 8.4Hs|NM_033360.2|5213 5213 4451 1 47.6 3.6 42.6 5.1 84.6 6.4 66.6 8.0Hs|NM_033360.2|5214 5214 4452 1 43.5 5.2 42.7 9.1 106.3 1.7 82.9 2.2Hs|NM_033360.2|5234 5234 4472 1 45.0 3.8 47.3 5.0 103.1 4.9 83.9 4.0Hs|NM_033360.2|5235 5235 4473 1 39.8 9.1 41.1 7.7 86.6 13.1 71.4 10.3Hs|NM_033360.2|5252 5252 4491 1 34.4 17.6 39.8 17.3 139.6 6.9 127.6 9.9Hs|NM_033360.2|5253 5253 4492 1 24.2 12.4 23.9 11.2 Hs|NM_033360.2|52545254 4493 1 35.3 7.6 36.8 7.9 102.0 1.2 84.1 1.6 Hs|NM_033360.2|52555255 4494 1 38.1 7.3 32.2 4.5 71.0 3.3 86.8 1.7 Hs|NM_033360.2|5256 52564497 1 52.5 5.8 48.3 7.2 89.4 2.2 93.8 4.1 Hs|NM_033360.2|5257 5257 45421 40.0 7.6 36.6 8.8 90.3 5.7 79.7 4.6 Hs|NM_033360.2|5258 5258 4548 127.1 8.7 21.1 6.4 90.8 3.0 86.0 4.0 Hs|NM_033360.2|5259 5259 4551 1 44.720.6 50.5 24.9 124.7 4.2 102.6 3.0 Hs|NM_033360.2|5260 5260 4591 1 48.02.0 50.1 2.8 110.2 3.6 108.4 3.3 Hs|NM_033360.2|5299 5299 4594 1 58.73.4 64.4 3.3 148.0 7.0 131.7 9.8 Hs|NM_033360.2|5300 5300 4597 1 87.69.8 93.5 6.4 127.6 11.6 136.8 10.6 Hs|NM_033360.2|5304 5304 4495 1 116.37.7 91.4 8.9 91.2 2.0 107.3 2.7 Hs|NM_033360.2|5305 5305 4498 1 107.06.1 109.6 6.2 117.0 5.4 126.7 4.5 Hs|NM_033360.2|5306 5306 4543 1 97.61.4 110.9 1.7 99.0 8.4 95.3 7.2 Hs|NM_033360.2|5307 5307 4549 1 98.3 5.2113.5 1.6 116.9 2.1 112.7 1.8 Hs|NM_033360.2|5308 5308 4552 1 147.8 11.0180.2 11.4 143.6 10.3 124.2 7.3 Hs|NM_033360.2|5309 5309 4592 1 129.48.9 152.9 11.7 118.2 5.2 115.1 6.7 Hs|NM_033360.2|5347 5347 4595 1 41.52.4 45.4 3.9 105.0 9.6 90.0 7.1 Hs|NM_033360.2|5348 5348 4632 1 75.8 8.988.4 6.6 119.6 8.6 105.3 12.9 Hs|NM_033360.2|5349 5349 4496 1 78.2 2.765.1 3.0 95.2 3.2 111.7 1.9 Hs|NM_033360.2|5350 5350 4499 1 92.3 2.794.9 2.5 114.7 3.0 125.1 2.7 Hs|NM_033360.2|5351 5351 4547 1 61.8 13.767.0 16.6 96.1 5.2 81.8 9.0 Hs|NM_033360.2|5352 5352 4550 1 54.8 11.258.5 6.0 97.8 6.1 93.4 6.1 Hs|NM_033360.2|5353 5353 4590 1 75.5 8.8 89.79.5 124.3 9.8 108.1 8.3 Hs|NM_033360.2|5354 5354 4593 1 52.7 16.3 63.216.3 90.4 4.4 85.8 6.9 Hs|NM_033360.2|5389 5389 4596 1 44.4 3.1 52.5 4.7110.6 5.2 98.3 2.7 Hs|NM_033360.2|5390 5390 4633 1 36.7 5.0 36.1 6.397.8 2.4 90.8 2.8 Hs|NM_033360.2|5391 5391 4634 1 29.0 5.7 26.4 5.9 62.73.5 67.1 5.6 Hs|NM_033360.2|5392 5392 4635 1 45.8 5.2 44.9 4.5 98.7 6.795.3 8.4 Hs|NM_033360.2|5393 5393 4636 1 51.3 3.6 52.7 4.1 110.4 2.3101.9 1.9As shown in Table 9, 103 of 243 asymmetric DsiRNAs examined showedgreater than 70% reduction of human KRAS levels in HeLa cells at 1 nM,in both qPCR assays used to determine human KRAS levels. Of these 103DsiRNAs, 37 exhibited greater than 80% reduction of human KRAS levels inHeLa cells at 1 nM in both qPCR assays used to determine human KRASlevels. A number of asymmetric DsiRNAs also capable of inhibiting mouseKRAS levels in mouse Hepa 1-6 cells at 1 nM in the environment of a cellwere also identified in such assays.

120 asymmetric DsiRNAs of the above experiment were then examined in asecondary assay (“Phase 2”), with results of such assays presented inhistogram form in FIGS. 12-31. Specifically, the 120 asymmetric DsiRNAsselected from the 243 tested above were assessed for inhibition of humanKRAS at 1 nM, 0.1 nM and 0.1 nM (0.1 nM was performed in duplicate,independent assays for each KRAS-targeting asymmetric DsiRNA) in theenvironment of human HeLa cells (FIGS. 12-21). These 120 asymmetricDsiRNAs were also assessed for inhibition of mouse KRAS at 1 nM, 0.1 nMand 0.1 nM (again, 0.1 nM was performed in duplicate, independent assaysfor each KRAS-targeting asymmetric DsiRNA) in the environment of mouseHepa 1-6 cells (FIGS. 22-31). As shown in FIGS. 12-21, a remarkablenumber of asymmetric DsiRNAs reproducibly exhibited robust human KRASinhibitory efficacies at 100 pM when assayed in the environment of HeLacells. In addition, as shown in FIGS. 22-31, a number of theseasymmetric DsiRNAs also showed robust mouse KRAS inhibitory efficaciesat 1 nM and 100 pM when assayed in the environment of mouse Hepa 1-6cells. (Meanwhile, human KRAS-specific inhibitory asymmetric DsiRNAswere also identified.)

Example 7 Inhibition of KRAS by Additional Preferred DsiRNAs

The remaining DsiRNA molecules shown in Tables 4-5 possessing sense andantisense strand sequences as shown and targeting KRAS wild-typesequences (and variant sequences where applicable) are designed andsynthesized as described above and tested in HeLa cells (and,optionally, in mouse Hepa 1-6 cells) for inhibitory efficacy asdescribed in Examples 3 and 6 above. The ability of these DsiRNA agentsto inhibit KRAS expression is optionally assessed in comparison tocorresponding KRAS target sequence-directed 21mer siRNAs (21mer siRNAidentification methods identical to those used to populate the siRNAsequences of Table 2 can be applied to the DsiRNAs of Tables 4-6). Theremaining selected DsiRNA agents of Tables 4-6 are expected to showefficacy as KRAS inhibitors, with a significant number of tested DsiRNAagents anticipated to exhibit greater than 50% reduction of the KRAStarget at 1 nM and at 100 pM. As seen for the DsiRNA agents of Tables2-3, a significant majority of DsiRNAs are anticipated to outperformcognate siRNA pairs, as determined via measurement of efficacy indecreasing levels of KRAS target relative to the cognate siRNA agent.The duration of such inhibitory effects is also examined at both 24hours and 48 hours post-administration, with concentrations of 0.1 nM, 1nM and 5 nM tested. As above, a significant majority of DsiRNAs isanticipated to outperform its cognate siRNA pair, as determined viameasurement of potency and duration of effect.

Example 8 Indications

The present body of knowledge in KRAS research indicates the need formethods to assay KRAS activity and for compounds that can regulate KRASexpression for research, diagnostic, and therapeutic use. As describedherein, the nucleic acid molecules of the present invention can be usedin assays to diagnose disease state related to KRAS levels. In addition,the nucleic acid molecules can be used to treat disease state related toKRAS misregulation, levels, etc.

Particular disorders and disease states that can be associated with KRASexpression modulation include, but are not limited to cancer and/orproliferative diseases, conditions, or disorders and other diseases,conditions or disorders that are related to or will respond to thelevels of KRAS in a cell or tissue, alone or in combination with othertherapies. Particular degenerative and disease states that areassociated with KRAS expression modulation include but are not limitedto, for example lung cancer, colorectal cancer, bladder cancer,pancreatic cancer, breast cancer, and prostate cancer.

Gemcytabine and cyclophosphamide are non-limiting examples ofchemotherapeutic agents that can be combined with or used in conjunctionwith the nucleic acid molecules (e.g. DsiRNA molecules) of the instantinvention. Those skilled in the art will recognize that other drugs suchas anti-cancer compounds and therapies can be similarly be readilycombined with the nucleic acid molecules of the instant invention (e.g.DsiRNA molecules) and are hence within the scope of the instantinvention. Such compounds and therapies are well known in the art (seefor example Cancer: Principles and Practice of Oncology, Volumes 1 and2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J. B.Lippincott Company, Philadelphia, USA) and include, without limitations,antifolates; fluoropyrimidines; cytarabine; purine analogs; adenosineanalogs; amsacrine; topoisomerase I inhibitors; anthrapyrazoles;retinoids; antibiotics such as bleomycin, anthacyclins, mitomycin C,dactinomycin, and mithramycin; hexamethylmelamine; dacarbazine;1-asperginase; platinum analogs; alkylating agents such as nitrogenmustard, melphalan, chlorambucil, busulfan, ifosfamide,4-hydroperoxycyclophosphamide, nitrosoureas, thiotepa; plant derivedcompounds such as vinca alkaloids, epipodophyllotoxins, taxol;Tamoxifen; radiation therapy; surgery; nutritional supplements; genetherapy; radiotherapy such as 3D-CRT; immunotoxin therapy such as ricin,monoclonal antibodies Herceptin; and the like. For combination therapy,the nucleic acids of the invention are prepared in one of two ways.First, the agents are physically combined in a preparation of nucleicacid and chemotherapeutic agent, such as a mixture of a nucleic acid ofthe invention encapsulated in liposomes and ifosfamide in a solution forintravenous administration, wherein both agents are present in atherapeutically effective concentration (e.g., ifosfamide in solution todeliver 1000-1250 mg/m2/day and liposome-associated nucleic acid of theinvention in the same solution to deliver 0.1-100 mg/kg/day).Alternatively, the agents are administered separately but simultaneouslyin their respective effective doses (e.g., 1000-1250 mg/m2/d ifosfamideand 0.1 to 100 mg/kg/day nucleic acid of the invention).

Those skilled in the art will recognize that other compounds andtherapies used to treat the diseases and conditions described herein cansimilarly be combined with the nucleic acid molecules of the instantinvention (e.g. siNA molecules) and are hence within the scope of theinstant invention.

Example 9 Serum Stability for DsiRNAs

Serum stability of DsiRNA agents is assessed via incubation of DsiRNAagents in 50% fetal bovine serum for various periods of time (up to 24h) at 37° C. Serum is extracted and the nucleic acids are separated on a20% non-denaturing PAGE and visualized with Gelstar stain. Relativelevels of protection from nuclease degradation are assessed for DsiRNAs(optionally with and without modifications).

Example 10 Use of Additional Cell Culture Models to Evaluate theDown-Regulation of KRAS Gene Expression

A variety of endpoints have been used in cell culture models to look atRas-mediated effects after treatment with anti-Ras agents. Phenotypicendpoints include inhibition of cell proliferation, RNA expression, andreduction of Ras protein expression. Because KRAS oncogene mutations aredirectly associated with increased proliferation of certain tumor cells,a proliferation endpoint for cell culture assays is preferably used asthe primary screen. There are several methods by which this endpoint canbe measured. Following treatment of cells with DsiRNA, cells are allowedto grow (typically 5 days), after which the cell viability, theincorporation of [³H] thymidine into cellular DNA and/or the celldensity are measured. The assay of cell density can be done in a 96-wellformat using commercially available fluorescent nucleic acid stains(such as Syto® 13 or CyQuant®). As a secondary, confirmatory endpoint, aDsiRNA-mediated decrease in the level of KRas protein expression can beevaluated using a KRas-specific ELISA.

Example 11 Evaluation of Anti-KRAS DsiRNA Efficacy in a Mouse Model ofKRAS Misregulation

Anti-KRAS DsiRNA chosen from in vitro assays can be further tested inmouse models, including, e.g., xenograft and other animal models asrecited above. In one example, mice possessing misregulated (e.g.,elevated) KRAS levels are administered a DsiRNA agent of the presentinvention via hydrodynamic tail vein injection. 3-4 mice per group(divided based upon specific DsiRNA agent tested) are injected with 50μg or 200 μg of DsiRNA. Levels of KRAS RNA are evaluated using RT-qPCR.Additionally or alternatively, levels of KRas (e.g., KRas protein levelsand/or cancer cell/tumor formation, growth or spread) can be evaluatedusing an art-recognized method, or phenotypes associated withmisregulation of KRAS (e.g., tumor formation, growth, metastasis, etc.)are monitored (optionally as a proxy for measurement of KRAS transcriptor KRas protein levels). Active DsiRNA in such animal models can also besubsequently tested in combination with standard chemotherapies.

Example 12 Diagnostic Uses

The DsiRNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of DsiRNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. DsiRNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells. The close relationship between DsiRNA activityand the structure of the target KRAS RNA allows the detection ofmutations in a region of the KRAS molecule, which alters thebase-pairing and three-dimensional structure of the target KRAS RNA. Byusing multiple DsiRNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target KRAS RNAswith DsiRNA molecules can be used to inhibit gene expression and definethe role of specified gene products in the progression of aKRAS-associated disease or disorder. In this manner, other genetictargets can be defined as important mediators of the disease. Theseexperiments will lead to better treatment of the disease progression byaffording the possibility of combination therapies (e.g., multipleDsiRNA molecules targeted to different genes, DsiRNA molecules coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of DsiRNA molecules and/or other chemical or biologicalmolecules). Other in vitro uses of DsiRNA molecules of this inventionare well known in the art, and include detection of the presence of RNAsassociated with a disease or related condition. Such RNA is detected bydetermining the presence of a cleavage product after treatment with aDsiRNA using standard methodologies, for example, fluorescence resonanceemission transfer (FRET).

In a specific example, DsiRNA molecules that cleave only wild-type ormutant or polymorphic forms of the target KRAS RNA are used for theassay. The first DsiRNA molecules (i.e., those that cleave onlywild-type forms of target KRAS RNA) are used to identify wild-type KRASRNA present in the sample and the second DsiRNA molecules (i.e., thosethat cleave only mutant or polymorphic forms of target RNA) are used toidentify mutant or polymorphic KRAS RNA in the sample. As reactioncontrols, synthetic substrates of both wild-type and mutant orpolymorphic KRAS RNA are cleaved by both DsiRNA molecules to demonstratethe relative DsiRNA efficiencies in the reactions and the absence ofcleavage of the “non-targeted” KRAS RNA species. The cleavage productsfrom the synthetic substrates also serve to generate size markers forthe analysis of wild-type and mutant KRAS RNAs in the sample population.Thus, each analysis requires two DsiRNA molecules, two substrates andone unknown sample, which is combined into six reactions. The presenceof cleavage products is determined using an RNase protection assay sothat full-length and cleavage fragments of each KRAS RNA can be analyzedin one lane of a polyacrylamide gel. It is not absolutely required toquantify the results to gain insight into the expression of mutant orpolymorphic KRAS RNAs and putative risk of KRAS-associated phenotypicchanges in target cells. The expression of KRAS mRNA whose proteinproduct is implicated in the development of the phenotype (i.e., diseaserelated/associated) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of KRAS RNA levels is adequate and decreases thecost of the initial diagnosis. Higher mutant or polymorphic form towild-type ratios are correlated with higher risk whether KRAS RNA levelsare compared qualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying DsiRNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08372816B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated double stranded nucleic acid (dsNA) comprising first andsecond nucleic acid strands and a duplex region of at least 25 basepairs, wherein each of said first and second nucleic acid strandscomprises RNA and has a length which is at least 25 and at most 35nucleotides, wherein said second oligonucleotide strand is sufficientlycomplementary to SEQ ID NO: 161 along at least 19 nucleotides of saidsecond oligonucleotide strand length to reduce KRAS target geneexpression when said double stranded nucleic acid is introduced into amammalian cell.
 2. The isolated dsNA of claim 1, wherein starting fromthe first nucleotide (position 1) at the 3′ terminus of the firstoligonucleotide strand, position 1, 2 and/or 3 is substituted with amodified nucleotide.
 3. The isolated dsNA of claim 2, wherein saidmodified nucleotide residue of said 3′ terminus of said first strand isselected from the group consisting of a deoxyribonucleotide, anacyclonucleotide and a fluorescent molecule.
 4. The isolated dsNA ofclaim 2, wherein position 1 of said 3′ terminus of the firstoligonucleotide strand is a deoxyribonucleotide.
 5. The isolated dsNA ofclaim 1, wherein said 3′ terminus of said first strand and said 5′terminus of said second strand form a blunt end.
 6. The isolated dsNA ofclaim 1, wherein said first strand is 25 nucleotides in length and saidsecond strand is 27 nucleotides in length.
 7. The isolated dsNA of claim1, wherein said second strand comprises SEQ ID NO:
 26. 8. The isolateddsNA of claim 1, wherein said first strand comprises SEQ ID NO:
 105. 9.The isolated dsNA of claim 1 comprising a first strand sequencecomprising SEQ ID NO: 105 and a second strand sequence comprising SEQ IDNO:
 26. 10. The isolated dsNA of claim 1, wherein each of said first andsaid second strands has a length which is at least 26 nucleotides. 11.The isolated dsNA of claim 1, wherein all nucleotides of said 1-5single-stranded nucleotides at said 3′ terminus of said second strandare modified nucleotides.
 12. The isolated dsNA of claim 1, wherein oneor both of the first and second oligonucleotide strands comprises a 5′phosphate.
 13. The isolated dsNA of claim 1, wherein said secondoligonucleotide strand, starting from the nucleotide residue of saidsecond strand that is complementary to the 5′ terminal nucleotideresidue of said first oligonucleotide strand, comprises alternatingmodified and unmodified nucleotide residues.
 14. The isolated dsNA ofclaim 1, wherein said second oligonucleotide strand comprises anucleotide residue that forms a mismatch with SEQ ID NO:
 161. 15. Theisolated dsNA of claim 1, wherein each of said first and said secondstrands has a length which is at least 26 and at most 30 nucleotides.16. The isolated dsNA of claim 1, wherein said dsRNA is cleavedendogenously in said cell by Dicer.
 17. The isolated dsNA of claim 1,wherein the amount of said isolated double stranded nucleic acidsufficient to reduce expression of the target gene is selected from thegroup consisting of 1 nanomolar or less, 200 picomolar or less, 100picomolar or less, 50 picomolar or less, 20 picomolar or less, 10picomolar or less, 5 picomolar or less, 2, picomolar or less and 1picomolar or less in the environment of said cell.
 18. The isolated dsNAof claim 1, wherein said isolated dsRNA possesses greater potency thanan isolated 21 mer siRNA directed to the identical at least 19nucleotides of SEQ ID NO: 161 in reducing target KRAS gene expressionwhen assayed in vitro in a mammalian cell at an effective concentrationin the environment of a cell of 1 nanomolar or less.
 19. The isolateddsNA of claim 1, wherein said isolated dsRNA possesses greater potencythan an isolated 21 mer siRNA directed to the identical at least 19nucleotides of SEQ ID NO: 161 in reducing target KRAS gene expressionwhen assayed in vitro in a mammalian cell at an effective concentrationin the environment of a cell selected from the group consisting of 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, 10 picomolar or less, 5 picomolar or less, 2,picomolar or less and 1 picomolar or less in the environment of saidcell.
 20. The isolated dsNA of claim 1, wherein said isolated dsNA issufficiently complementary to SEQ ID NO: 161 to reduce KRAS target geneexpression by an amount (expressed by %) selected from the groupconsisting of at least 10%, at least 50%, at least 80-90%, at least 95%,at least 98%, and at least 99% when said double stranded nucleic acid isintroduced into a mammalian cell.
 21. The isolated dsNA of claim 1,wherein the first and second strands are joined by a chemical linker.22. The isolated double stranded nucleic acid of claim 1, wherein said3′ terminus of said first strand and said 5′ terminus of said secondstrand are joined by a chemical linker.
 23. The isolated double strandednucleic acid of claim 1, wherein a nucleotide of said second or firststrand is substituted with a modified nucleotide that directs theorientation of Dicer cleavage.
 24. The isolated double stranded nucleicacid of claim 1 comprising a modified nucleotide selected from the groupconsisting of a deoxyribonucleotide, a dideoxyribonucleotide, anacyclonucleotide, a 3′-deoxyadenosine (cordycepin), a3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a2′,3′-dideoxy-3′-thiacytidine (3TC), a2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotideof 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine(3TC) and a monophosphate nucleotide of2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkylribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide,a 2′-fluoro ribonucleotide, and a locked nucleic acid.
 25. The isolateddouble stranded nucleic acid of claim 1 comprising a phosphate backbonemodification selected from the group consisting of a phosphonate, aphosphorothioate and a phosphotriester.
 26. The isolated double-strandednucleic acid of claim 1, wherein said second strand of said dsNAcomprises 1-5 single-stranded nucleotides at its 3′ terminus.
 27. Theisolated dsNA of claim 26, wherein said nucleotides of said 1-5single-stranded nucleotides at said 3′ terminus of said second strandcomprise a modified nucleotide.
 28. The isolated dsNA of claim 27,wherein said modified nucleotide of said 1-5 single-stranded nucleotidesat said 3′ terminus of said second strand is a 2′-O-methylribonucleotide.
 29. The isolated dsNA of claim 27 wherein said modifiednucleotide residues are selected from the group consisting of2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O-(N-methlycarbamate).30. The isolated dsNA of claim 27 comprising two single-strandednucleotides at said 3′ terminus of said second strand, wherein said twosingle-stranded nucleotides comprise a 2′-O-methyl modifiedribonucleotide.
 31. The isolated dsNA of claim 26 comprising 1-3single-stranded nucleotides at said 3′ terminus of said second strand.32. The isolated dsNA of claim 26 comprising 1-2 single-strandednucleotides at said 3′ terminus of said second strand.
 33. The isolateddouble-stranded nucleic acid of claim 1, wherein said secondoligonucleotide strand is sufficiently complementary to SEQ ID NO: 6863along at least 19 nucleotides of said second oligonucleotide strandlength to reduce KRAS target gene expression when said double strandednucleic acid is introduced into a mammalian cell.
 34. The isolateddouble-stranded nucleic acid of claim 1, wherein said secondoligonucleotide strand comprises a sequence that is completelycomplementary to SEQ ID NO:
 6863. 35. A mammalian cell containing theisolated dsNA of claim
 1. 36. A pharmaceutical composition comprisingthe isolated dsNA of claim 1 and a pharmaceutically acceptable carrier.37. A kit comprising the isolated dsNA of claim 1 and instructions forits use.
 38. A composition possessing KRAS inhibitory activityconsisting essentially of an isolated double stranded ribonucleic acid(dsNA) comprising first and second nucleic acid strands and a duplexregion of at least 25 base pairs, wherein said second strand of saiddsNA comprises 1-5 single-stranded nucleotides at its 3′ terminus,wherein said second oligonucleotide strand is sufficiently complementaryto SEQ ID NO: 161 along at least 19 nucleotides of said secondoligonucleotide strand length to reduce KRAS target gene expression whensaid double stranded nucleic acid is introduced into a mammalian cell.39. A method for reducing expression of a target KRAS gene in amammalian cell comprising contacting a mammalian cell in vitro with anisolated dsNA of claim 1 in an amount sufficient to reduce expression ofa target KRAS gene in said cell.
 40. The method of claim 39, whereintarget KRAS gene expression is reduced by an amount (expressed by %)selected from the group consisting of at least 10%, at least 50% and atleast 80-90%.
 41. The method of claim 39, wherein KRAS mRNA levels arereduced by an amount (expressed by %) of at least 90% at least 8 daysafter said cell is contacted with said dsNA.
 42. The method of claim 39,wherein KRAS mRNA levels are reduced by an amount (expressed by %) of atleast 70% at least 10 days after said cell is contacted with said dsNA.43. A method for reducing expression of a target KRAS gene in a mammalcomprising administering an isolated dsNA of claim 1 to a mammal in anamount sufficient to reduce expression of a target KRAS gene in themammal.
 44. The method of claim 43, wherein said isolated dsNA isadministered at a dosage selected from the group consisting of 1microgram to 5 milligrams per kilogram of said mammal per day, 100micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams perkilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms perkilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 microgramsper kilogram.
 45. The method of claim 43, wherein said isolated dsNApossesses greater potency than an isolated 21 mer siRNA directed to theidentical at least 19 nucleotides of SEQ ID NO: 161 in reducing targetKRAS gene expression when assayed in vitro in a mammalian cell at aneffective concentration in the environment of a cell of 1 nanomolar orless.
 46. The method of claim 43, wherein said administering stepcomprises a mode selected from the group consisting of intravenousinjection, intramuscular injection, intraperitoneal injection, infusion,subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical,oral and inhaled delivery.
 47. A method for selectively inhibiting thegrowth of a cell comprising contacting a cell with an amount of anisolated dsNA of claim 1 sufficient to inhibit the growth of the cell.48. The method of claim 47, wherein said cell is a tumor cell of asubject.
 49. The method of claim 47, wherein said cell is a tumor cellin vitro.
 50. The method of claim 47, wherein said cell is a human cell.51. A formulation comprising the isolated dsNA of claim 1, wherein saiddsNA is present in an amount effective to reduce target KRAS RNA levelswhen said dsNA is introduced into a mammalian cell in vitro by an amount(expressed by %) selected from the group consisting of at least 10%, atleast 50% and at least 80-90%, and wherein said dsNA possesses greaterpotency than an isolated 21 mer siRNA directed to the identical at least19 nucleotides of SEQ ID NO: 161 in reducing target KRAS RNA levels whenassayed in vitro in a mammalian cell at an effective concentration inthe environment of a cell of 1 nanomolar or less.
 52. The formulation ofclaim 51, wherein said effective amount is selected from the groupconsisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolaror less, 50 picomolar or less, 20 picomolar or less, 10 picomolar orless, 5 picomolar or less, 2, picomolar or less and 1 picomolar or lessin the environment of said cell.
 53. A formulation comprising theisolated dsNA of claim 1, wherein said dsNA is present in an amounteffective to reduce target RNA levels when said dsNA is introduced intoa cell of a mammalian subject by an amount (expressed by %) selectedfrom the group consisting of at least 10%, at least 50% and at least80-90%, and wherein said dsNA possesses greater potency than an isolated21 mer siRNA directed to the identical at least 19 nucleotides of SEQ IDNO: 161 in reducing target KRAS RNA levels when assayed in vitro in amammalian cell at an effective concentration in the environment of acell of 1 nanomolar or less.
 54. The formulation of claim 53, whereinsaid effective amount is a dosage selected from the group consisting of1 microgram to 5 milligrams per kilogram of said subject per day, 100micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams perkilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms perkilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 microgramsper kilogram.
 55. The method of claim 47, wherein said cell is apancreatic carcinoma cell.
 56. The formulation of claim 51, wherein saidformulation comprises a lipid.
 57. An isolated double stranded nucleicacid (dsNA) comprising first and second nucleic acid strands, whereineach of said first and second nucleic acid strands comprises RNA,wherein said second oligonucleotide strand is 19 to 35 nucleotides inlength and comprises a sequence that is completely complementary to SEQID NO: 6863 and wherein said dsNA reduces KRAS target gene expressionwhen said double stranded nucleic acid is introduced into a mammaliancell.